Methods of enriching for and identifying polymorphisms

ABSTRACT

The invention encompasses methods for enriching for and identifying a polymorphism within a nucleic acid sample either by separating a subset of a nucleic acid sample or by selectively replicating a subset of a nucleic acid sample such that the polymorphism is contained within a nucleic acid population with reduced complexity, and then identifying the polymorphism within the enriched nucleic acid sample. Methods also are disclosed for enriching for and identifying a polymorphism by contacting a nucleic acid sample that includes a subset of nucleic acid molecules having a sequence that binds to a sequence-specific binding activity with a molecule having a sequence-specific binding activity under conditions which permit specific binding, such that the subset of nucleic acid molecules bound to the activity is enriched for nucleic acid molecules having the sequence recognized by the sequence-specific binding activity, and detecting a polymorphism with respect to a reference sequence in the subset of nucleic acid molecules.

FIELD OF THE INVENTION

The present invention relates in general to nucleic acid sequenceanalysis, and in particular to methods which facilitate theidentification of sequence polymorphisms.

BACKGROUND OF THE INVENTION

Genomic amplification strategies using the polymerase chain reaction(PCR; Mullis & Faloona, 1987, Meth. Enzymol. 155:335) are employed tofacilitate the identification of polymorphic sequences. PCR is used toamplify regions of genomic DNA that carry potential polymorphisms. Onemethod hybridizes the PCR products to allele-specific hybridizationprobes (Saiki et al., 1986, Nature 324:163). Other methods utilizeoligonucleotide primers that either match or mismatch the targetedpolymorphism (Newton et al., 1989, Nucleic Acids Res. 17:2503).

With methods that hybridize the PCR product to an allele-specific probe,PCR is used to reduce the complexity of the DNA sample being assayed forthe polymorphic marker and to increase the number of copies of thepolymorphism-bearing DNA. If 100,000 polymorphic markers were to beassayed per genome, it would be very expensive to perform 100,000individual PCR reactions. Some advances have been made to multiplex PCRreactions (Chamberlain et al., 1988, Nucl. Acids Res. 16: 11141), andthe degree of multiplexing of the PCR has been scaled up, followed byhybridization to an array of allele-specific probes (Wang et al., 1998,Science 280: 1077). However, in the studies by Wang et al., thepercentage of PCR products that successfully amplified decreased as thenumber of PCR primers added to the reaction increased. Whenapproximately 100 primer pairs were used, about 90% of the PCR productswere successfully amplified. When the number of primer pairs wasincreased to about 500, about 50% of the PCR products were successfullyamplified. Another disadvantage with multiplex PCR is that individualprimer pairs must be synthesized for each polymorphic target. GenotypingDNA with 100,000 polymorphism targets would require, in theory, 200,000different PCR primers. Not only is the synthesis of such primers costlyand time consuming, but not all primer designs succeed in producing adesired PCR product. Therefore considerable time and energy may be spentoptimizing the primer designs.

Hatada et al. have cleaved genomic DNA with a rarely cutting restrictionenzyme, separated the cleaved DNA by gel electrophoresis, again cleavedthe separated DNA with a second restriction enzyme in the gel, and againseparated the DNA in a second dimension by electrophoresis (Hatada etal., 1991, Proc. Natl. Acad. Sci. USA 88: 9523). According to the Hatadaet al. method, one then examines the two-dimensional pattern of DNAspots using DNA from different individuals. Differences in DNA migrationpatterns result from sequence or nucleotide methylation differences inthe restriction enzyme recognition sequences.

Hayashizaki et al. (Hayashizaki et al., 1992, Genomics 14:733) usesolid-phase adapters specific for restriction fragment ends tophysically separate a subset of fragments from genomic DNA. Afterpurification of the adapter-bound DNA fraction away from the rest of thegenomic DNA, the bound DNA is separated from the adapters by cleavingagain with the restriction enzyme used for the adapter ligation. The DNAreleased from the adapters is then cloned into a replication vector tomake a gene library.

Others have used DNA binding factors to reduce the complexity ofpopulations of synthetic oligonucleotides with stretches of randomizedsequences, with the aim of elucidating the consensus binding sequencesof the proteins (Mavrothalassitis et al., 1990, DNA Cell Biol., 9:783;Blackwell & Weintraub, 1990, Science, 250: 1104; Woodring et al., 1993,Trends Biol. Sci. 18: 77, Hardenbol & Van Dyke, 1996, Proc. Natl. Acad.Sci. U.S.A., 93: 2811).

There is a need in the art for improved methods of identifyingpolymorphic sequences.

SUMMARY

The invention encompasses a method of enriching for and identifying anucleic acid sequence difference with respect to a reference sequencecomprising: a) contacting a nucleic acid sample with a moleculecomprising a sequence-specific binding activity under conditions whichpermit specific binding, wherein the sample comprises a subset ofnucleic acid molecules having a sequence that binds to thesequence-specific binding activity, and wherein a bound subset ofnucleic acid molecules is retained by the sequence-specific bindingactivity, such that the subset of bound nucleic acid molecules isenriched for molecules comprising the sequence recognized by thesequence-specific binding activity; and b) detecting a sequencedifference with respect to a reference sequence in the subset of nucleicacid molecules.

In a preferred embodiment of the invention, the molecule comprisingsequence-specific binding activity is selected from the group consistingof: transcription factors or DNA binding domains thereof; proteins withzinc-finger DNA binding domains: restriction endonuclease DNArecognition domains; sequence-specific antibodies; oligonucleotidescomplementary to an adapter ligated to a population of DNA molecules;nucleic acid molecules; aptamers; peptide nucleic acid molecules;peptides; and affinity resins which recognize DNA having a particularG+C content or methylation status.

In a preferred embodiment of the invention, the sequence-specificbinding activity is bound to a solid support.

The invention also encompasses a method of identifying nucleic acidsequence differences with respect to a reference sequence comprising: a)cleaving a nucleic acid sample from one or more individuals with one ormore sequence-specific cleavage agents to produce nucleic acidfragments; b) operatively linking the fragments of step (a) withmolecules capable of being replicated; c) introducing the linkedmolecules of step (b) into a system capable of replicating only a subsetof the linked molecules, and replicating the subset to form a collectionof replicated molecules; and d) detecting one or more nucleic acidsequence differences with respect to a reference sequence in the membersof the collection of step (c) with a method capable of detecting one ormore nucleotide differences with respect to a reference sequence.

In a preferred embodiment, the system capable of replicating the linkedmolecules comprises host cells and the collection of replicatedmolecules comprises a library.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises DNA sequencing.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises denaturing HPLC.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises electrophoresis capable of detectingconformational differences in the nucleic acids.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises a protein capable of detectingmismatches between duplexed strands of nucleic acid.

In a preferred embodiment, the sequencing is performed using primersthat hybridize to the molecules capable of being replicated.

In a preferred embodiment, the system capable of replicating the linkedmolecules comprises in vitro replication of the linked molecules.

In a preferred embodiment, the in vitro replication comprises a steputilizing primers for nucleic acid polymerization that hybridizespecifically to the molecules capable of being replicated.

In a preferred embodiment, the in vitro replication comprises a steputilizing primers for nucleic acid polymerization that hybridizespecifically to sequences comprising both a segment of the moleculescapable of being replicated and the fragment ends of a subset of thenucleic acid molecules linked to the molecules capable of beingreplicated.

In a preferred embodiment, the one or more cleavage agents may be one ormore restriction endonucleases. It is preferred that at least one of therestriction endonuclease cleaves DNA infrequently.

In a preferred embodiment, the infrequently cleaving restrictionendonuclease is selected from the group consisting of AscI, BssHII,EagI, NheI, NotI, PacI, PmeI, RsrII, SalI, SbfI, SfiI, SgrAI, SpeI,SrfI, and SwaI restriction endonucleases.

The invention also encompasses a method of identifying nucleic acidsequence differences with respect to a reference sequence comprising: a)cleaving a nucleic acid sample from one or more individuals with one ormore sequence-specific cleavage agents to produce nucleic acidfragments, wherein the ends of only a subset of the fragments comprisesequences capable of being operatively linked to a separation element;b) operatively linking the subset of step (a) with the separationelement; c) separating the linked molecules; and d) detecting one ormore nucleic acid sequence differences with respect to a referencesequence in the members of the separated molecules of step (c) with amethod capable of detecting one or more nucleotide differences withrespect to a reference sequence.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises DNA sequencing.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises denaturing HPLC.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises electrophoresis capable of detectingconformational differences in the nucleic acids.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises a protein capable of detectingmismatches between duplexed strands of nucleic acid.

In a preferred embodiment, the sequencing is performed using primersthat hybridize to the sequences capable of being operatively linked to aseparation element. In a preferred embodiment, the one or more cleavageagents are one or more restriction endonucleases. It is preferred thatat least one restriction endonuclease cleaves DNA infrequently.

In a preferred embodiment, the infrequently cleaving restrictionendonuclease is selected from the group consisting of AscI, BssHII,EagI, NheI, NotI, PacI, PmeI, RsrII. SalI, SbfI, SfiI, SgrAI, SpeI,SrfI, and SwaI restriction endonucleases.

The invention also encompasses a method of enriching for and identifyingnucleic acid sequence differences with respect to a reference sequencecomprising: a) fragmenting a nucleic acid sample from one or moreindividuals to an average fragment length; b) physically separating asubset of the nucleic acid fragments generated in step (a) based on thepresence or absence of a particular nucleotide sequence within thefragments: c) operatively linking the subset of step (b) with moleculescapable of being replicated; d) introducing the linked molecules of step(c) into a system capable of replicating the linked molecules, andreplicating the linked molecules to form a collection of replicatedmolecules, and e) detecting a nucleic acid sequence difference withrespect to a reference sequence in the collection of replicatedmolecules of step (d) using a method capable of detecting one or morenucleotide differences with respect to a reference sequence.

In a preferred embodiment, the system capable of replicating the linkedmolecules comprises host cells and the collection of replicatedmolecules comprises a library.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises DNA sequencing.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises denaturing HPLC.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises electrophoresis capable of detectingconformational differences in the nucleic acids.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises a protein capable of detectingmismatches between duplexed strands of nucleic acid.

In a preferred embodiment, the DNA sequencing is performed using primersthat hybridize to the molecules capable of being replicated.

In a preferred embodiment, the system capable of replicating the linkedmolecules comprises in vitro replication of the linked molecules.

In a preferred embodiment, the in vitro replication comprises a steputilizing primers for nucleic acid polymerization that hybridizespecifically to the molecules capable of being replicated.

In a preferred embodiment, the in vitro replication comprises a steputilizing primers for nucleic acid polymerization that hybridizespecifically to sequences comprising both a segment of the moleculescapable of being replicated and the fragment ends of a subset of thenucleic acid molecules linked to the molecules capable of beingreplicated.

In a preferred embodiment, the in vitro replication is repeated one ormore times to increase the enrichment of the linked molecules.

In a preferred embodiment, the method used to physically separate asubset of fragments comprises using a sequence-specific bindingmolecule.

In a preferred embodiment, the sequence-specific binding molecule is aprotein.

In a preferred embodiment, the one or more cleavage agents arerestriction endonucleases.

The invention also encompasses a method of enriching for and identifyingnucleic acid sequence differences with respect to a reference sequencecomprising: a) fragmenting a nucleic acid sample from one or moreindividuals to an average fragment length; b) separating a subset of thenucleic acid fragments based on the presence or absence of a nucleotidesequence within the fragments; c) detecting one or more nucleic acidsequence differences with respect to a reference sequence in the membersof the separated molecules of step (b) with a method capable ofdetecting one or more nucleotide differences with respect to a referencesequence.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises DNA sequencing.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises denaturing HPLC.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises electrophoresis capable of detectingconformational differences in the nucleic acids.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises a protein capable of detectingmismatches between duplexed strands of nucleic acid.

In a preferred embodiment, the DNA sequencing is performed using primersthat hybridize to the molecules capable of being replicated.

In a preferred embodiment, the method used to physically separate asubset of fragments comprises using a sequence-specific bindingmolecule.

In a preferred embodiment, the sequence-specific binding molecule is aprotein.

The invention also encompasses a method of enriching for and identifyingnucleic acid sequence differences with respect to a reference sequencecomprising: a) hybridizing a nucleic acid sample from one or moreindividuals with oligonucleotide primers under conditions wherein eachof the primers permits extension by a polymerase of two or moredifferent sequences, and wherein the sequences replicated by extensionof the primers comprise regions where there are known sequencedifferences between individuals of the species being examined; b)extending the oligonucleotide primers hybridized in step (a) to form anenriched collection of replicated molecules; and c) detecting one ormore nucleic acid sequence differences in the members of the collectionwith respect to a reference sequence with a method capable of detectingone or more nucleotide differences with respect to a reference sequence.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises DNA sequencing.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises denaturing HPLC.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises electrophoresis capable of detectingconformational differences in the nucleic acids.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises a protein capable of detectingmismatches between duplexed strands of nucleic acid.

In a preferred embodiment, the DNA sequencing is performed using primersthat hybridize to the primers hybridized in step (a) and extended instep (b).

In a preferred embodiment, steps (a) and (b) are repeated one or moretimes to increase the enrichment of the enriched collection ofreplicated molecules.

In a preferred embodiment, the method further comprises, after step (b)and before step (c) the step of hybridizing a second set of primers thathybridize specifically to sequences comprising both a segment of thefirst set of primers and a segment of the replicated portion of themolecules generated in step (b).

The invention also encompasses a method of enriching for and identifyingnucleic acid sequence differences with respect to a reference sequencecomprising: a) fragmenting a nucleic acid sample from one or moreindividuals; b) physically separating a subset of the nucleic acidfragments based on the size of the fragments: c) operatively linking thesubset of step (b) with molecules capable of being replicated; d)introducing the linked subset of molecules of step (c) into a systemcapable of replicating the linked subset of molecules, and replicatingthe subset of linked molecules to form an enriched collection ofreplicated molecules; and e) detecting one or more nucleotide sequencedifferences in the members of the collection of step (d) with a methodcapable of detecting one or more nucleotide differences with respect toa reference sequence.

In a preferred embodiment, the system capable of replicating the linkedmolecules comprises host cells and the collection of replicatedmolecules comprises a library.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises DNA sequencing.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises denaturing HPLC.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises electrophoresis capable of detectingconformational differences in the nucleic acids.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises a protein capable of detectingmismatches between duplexed strands of nucleic acid.

In a preferred embodiment, the sequencing is performed using primersthat hybridize to the molecules capable of being replicated.

In a preferred embodiment, the system capable of replicating the linkedmolecules comprises in vitro replication of the linked molecules.

In a preferred embodiment, the in vitro replication comprises a steputilizing primers for nucleic acid polymerization that hybridizespecifically to the molecules capable of being replicated.

In a preferred embodiment, the in vitro replication is repeated one ormore times to increase the enrichment of the collection of replicatedmolecules.

In a preferred embodiment, the in vitro replication comprises a steputilizing primers for nucleic acid polymerization that hybridizespecifically to sequences comprising both a segment of the moleculescapable of being replicated and the fragment ends of a subset of thenucleic acid molecules linked to the molecules capable of beingreplicated.

In a preferred embodiment, the physical separation by size of step (b)is accomplished using electrophoresis, density gradient centrifugation,or centrifugation through a viscous solution.

The invention also encompasses a method of enriching for and identifyingnucleic acid sequence differences with respect to a reference sequencecomprising: a) fragmenting a nucleic acid sample from one or moreindividuals; b) physically separating a subset of the nucleic acidfragments based on the size of the fragments; c) detecting one or morenucleic acid sequence differences with respect to a reference sequencein the members of the separated molecules of step (b) with a methodcapable of detecting one or more nucleotide differences with respect toa reference sequence.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises DNA sequencing.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises denaturing HPLC.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises electrophoresis capable of detectingconformational differences in the nucleic acids.

In a preferred embodiment, the method capable of detecting one or morenucleotide differences comprises a protein capable of detectingmismatches between duplexed strands of nucleic acid.

In a preferred embodiment, the physical separation by size isaccomplished using electrophoresis, density gradient centrifugation, orcentrifugation through a viscous solution.

The invention also encompasses a method for accessing a sub-portion of anucleic acid population, such method comprising: a) mixing one or moreoligonucleotide primers with a sample of the nucleic acid populationunder conditions which permit hybridization of one or more primers tothe sample, each primer comprising a 3′ terminal sequence whichhybridizes to an anchor sequence present in the nucleic acid sample; andwherein the one or more oligonucleotide primers contains an additional3′-terminal extension immediately adjacent to the sequence whichhybridizes to an anchor sequence; and b) adding ribonucleotides ordeoxynucleotides and a template-dependent polymerizing activity underconditions which permit extension of the one or more oligonucleotideprimers, such that the population of extended primers comprises asub-portion of nucleic acid molecules in the sample.

In a preferred embodiment, the primer comprises an additional3′-terminal extension immediately adjacent to the sequence whichhybridizes to an anchor sequence.

In a preferred embodiment, the additional 3′ terminal extension isselected from the group consisting of G, A, T and C.

In a preferred embodiment, the additional 3′ terminal extension isselected from the group consisting of: AA; AG; AC; AT; CA; CG; CC; CT;GA: GG; GC; GT; TA; TG; TC; and TT.

In a preferred embodiment, the additional 3′ terminal extension is atrinucleotide selected from the group consisting of: AAA; AAC; AAG; AAT; AGA; AGC; AGG; AGT; ACA; ACC; ACG; ACT; ATA; ATC; ATG; ATT; CAA; CAC;CAG; CAT; CCA; CCC; CCG; CCT; CGA; CGC; CGG; CGT; CTA; CTC; CTG; CTT;GAA; GAC; GAG; GAT; GCA; GCC; GCG; GCT; GGA; GGC; GGG; GGT; GTA; GTC;GTG; GTT; TAA; T AC; TAG; TAT; TCA; TCC; TCG; TCT; TGA; TGC; TGG; TGT;TTA; TTC; TTG; and TTT.

In a preferred embodiment, the additional 3′ terminal extension isselected from the group consisting of: tetranucleotides,pentanucleotides, hexanucleotides, septanucleotides, andoctanucleotides.

In a preferred embodiment, the anchor sequence is the recognitionsequence for a sequence-specific DNA binding activity selected from thegroup consisting of: transcription factors or DNA binding domainsthereof: proteins with zinc finger DNA-binding-domains; restrictionendonuclease DNA sequence recognition domains; sequence-specificantibodies; nucleic acid molecules; oligonucleotides complementary to anadapter ligated to a population of DNA molecules; aptamers; peptidenucleic acid molecules; peptides; and affinity resins which recognizeDNA having a particular G+C content or methylation status.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product tobetween about 500 and 5000 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 500 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 750 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 1000 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 1500 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 2000 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 3000 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 4000 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 5000 nucleotides in length.

In a preferred embodiment, the anchor sequence is a restrictionendonuclease recognition sequence.

In a preferred embodiment, the restriction endonuclease recognitionsequence occurs infrequently in the genome of the organism from whichthe nucleic acid sample is obtained.

In a preferred embodiment, the restriction endonuclease recognitionsequence that occurs infrequently in the genome of the organism fromwhich the nucleic acid sample is obtained is selected from the groupconsisting of: AscI, BssHII, EagI, NheI, NotI, PacI, PmeI, RsrII, SalI,SbfI, SfiI, SgrAI, SpeI, SrfI, and SwaI restriction endonucleaserecognition sequences.

In a preferred embodiment, one or more of the oligonucleotides ordeoxynucleotides is detectably labeled.

In a preferred embodiment, the label is selected from the groupconsisting of: fluorescent moieties; radioactive moieties; biotin; anddigoxigenin.

In a preferred embodiment, the oligonucleotide primer or primers is/areattached to a solid support or is/are labeled with a moiety allowingattachment to a solid support.

In a preferred embodiment, the method of accessing a sub-portion of anucleic acid population comprises the additional step of identifying anucleic acid sequence polymorphism in a population of individuals.

In a preferred embodiment, the method of accessing a sub-portion of anucleic acid population comprises the additional step of genotyping anindividual with respect to a nucleic acid sequence polymorphism.

The invention also encompasses a method for accessing a sub-populationof a genome, such method comprising: a) cleaving a nucleic acid samplewith a first restriction endonuclease wherein the recognition sequenceof the first restriction endonuclease occurs infrequently in the genome;b) ligating an adapter molecule to the cleaved ends generated in step(a), the adapter having an overhang complementary to that generated bythe first restriction endonuclease, and ligation of the adapter furtherfully or partially regenerating the recognition sequence of the firstrestriction endonuclease; c) mixing an oligonucleotide primercomplementary to the adapter molecule, wherein the 3′ terminus of theoligonucleotide primer is complementary to the fully or partiallyregenerated recognition sequence of the first restriction endonuclease,under conditions which permit hybridization of the oligonucleotideprimer to the adapter, and d) adding nucleotides and atemplate-dependent polymerizing activity under conditions which permitextension of the oligonucleotide primer, the resulting population ofprimer extension products comprising a sub-portion of the molecules inthe nucleic acid sample.

The invention also encompasses a method for accessing a sub-populationof a genome, such method comprising: a) cleaving a nucleic acid samplewith one or more cleavage agents to produce nucleic acid fragments; b)mixing one or more primers capable of annealing to nucleic acid fragmentends generated by the one or more cleavage agents and capable ofinitiating the replication of the nucleic acid regions comprising thefragment ends under conditions that permit the annealing; c) incubatingwith a polymerizing activity under conditions that permit extension ofthe one or more primers, the resulting population of primer extensionproducts comprising a sub-portion of the nucleic acid sequences in thegenome, wherein the sub-portion of the nucleic acid sequences comprisesan incomplete extension product.

In a preferred embodiment, the one or more cleavage agents aresequence-specific cleavage agents.

In a preferred embodiment, the one or more cleavage agents aresequence-specific cleavage agents and the primers comprise sequencescomplementary to the recognition sequence of the sequence-specificcleavage agents.

In a preferred embodiment, the primers additionally comprise 3′ endsequences capable of hybridizing to only a subset of the molecules inthe nucleic acid sample.

It is preferred that the 3′ end sequences comprise terminal extensionsimmediately adjacent to the sequence that hybridizes to the recognitionsequence;

It is also preferred that the terminal extensions are mononucleotidesselected from the group consisting of: A, C, G, and T.

It is also preferred that the extensions are dinucleotides selected fromthe group consisting of: AA; AG; AC; AT; CA; CC; CC; CT; GA; GG; GC; GT;TA; TG; TC; and TT.

It is also preferred that the extensions are trinucleotides selectedfrom the group consisting of: AAA; AAC; AAG; AA T; AGA; AGC; AGG; AGT;ACA; ACC; ACG; ACT; ATA; ATC; ATG; ATT; CAA; CAC; CAG; CAT; CCA; CCC;CCG; CCT; CGA; CGC; CGG; CGT; CTA; CTC; CTG; CTT; GAA; GAC; GAG; GAT;GCA; GCC; GCG; GCT; GGA; GGC; GGG; GGT; GTA; GTC; GTG; GTT; TAA; TAC;TAG; TAT; TCA; TCC; TCG; TCT; TGA; TGC; TGG; TGT; TTA; TTC; TTG; andTTT.

In a preferred embodiment, the extension is selected from the groupconsisting of: tetranucleotides, pentanucleotides, hexanucleotides,septanucleotides, and octanucleotides.

It should also be appreciated by one skilled in the art that the adaptermolecules that are operatively linked to the cleaved ends of nucleicacids may comprise a promoter sequence capable of initiating thesynthesis of RNA or DNA from the promoter site with an appropriatepolymerase. For example, the adapter may comprise a T7 RNA polymerasepromoter oriented so that transcription will proceed into the nucleicacid sample to which the adapter has been operatively linked.

The invention also encompasses a method for accessing a sub-populationof a genome, such method comprising: a) cleaving a nucleic acid samplewith one or more cleavage agents to produce nucleic acid fragments: b)operatively linking an adapter molecule to the cleaved ends generated instep (a); c) incubating with a polymerizing activity under conditionsthat permit nucleic acid synthesis from the adapter, the resultingpopulation of extension products comprising a sub-portion of the nucleicacid sequences in the genome, wherein the sub-portion of the nucleicacid sequences comprises an incomplete extension product.

In a preferred embodiment, the adapter molecule contains atranscriptional promoter.

In a preferred embodiment, the adapter molecule contains a free endcapable of being extended by a polymerizing activity.

In a preferred embodiment, the adapter molecule is double stranded andcontains a sequence capable of being nicked by a second cleavage agentto produce a free end capable of being extended by a polymerizingactivity.

The invention also encompasses a method for accessing a sub-populationof a genome, such method comprising: a) cleaving a nucleic acid samplewith one or more cleavage agents to produce nucleic acid fragments: b)operatively linking an adapter molecule to the cleaved ends generated instep (a); c) mixing a primer complementary to the adapter molecule withthe linked molecules generated in step (b) under conditions that permithybridization of the primer to the adapter; and d) incubating with apolymerizing activity under conditions that permit nucleic acidsynthesis from the adapter, the resulting population of primer extensionproducts comprising a sub-portion of the genome, wherein the sub-portionof the genome comprises an incomplete extension product.

In a preferred embodiment, the one or more cleavage agents aresequence-specific cleavage agents.

In a preferred embodiment, the one or more cleavage agents aresequence-specific cleavage agents and the primers comprise sequencescomplementary to the recognition sequence of the sequence-specificcleavage agents.

In a preferred embodiment, the primers additionally comprise 3′ endsequences capable of hybridizing to only a subset of the molecules inthe nucleic acid sample.

It is preferred that the 3′ end sequences comprise terminal extensionsimmediately adjacent to the sequence that hybridizes to the recognitionsequence.

It is also preferred that the terminal extensions are mononucleotidesselected from the group consisting of: A, C, G, and T.

It is also preferred that the extensions are dinucleotides selected fromthe group consisting of: AA; AG; AC; AT; CA; CG; CC; CT; GA; GG; GC; GT;TA; TG; TC; and TT.

It is also preferred that the extensions are trinucleotides selectedfrom the group consisting of: AAA; AAC; AAG; AA T; AGA; AGC; AGG; AGT;ACA; ACC; ACG; ACT; ATA; ATC; ATG; ATT; CAA; CAC; CAG; CAT; CCA; CCC;CCG; CCT; CGA; CGC; CGG; CGT; CTA; CTC; CTG; CTT; GAA; GAC; GAG; GAT;GCA; GCC; GCG; GCT; GGA; GGC; GGG; GGT; GTA; GTC; GTG; GTT; TAA; TAC;TAG; TAT; TCA; TCC; TCG; TCT; TGA; TGC; TGG; TGT; TTA; TTC; TTG; andTTT.

In a preferred embodiment, the extensions are selected from the groupconsisting of: tetranucleotides, pentanucleotides, hexanucleotides,septanucleotides, and octanucleotides.

The invention also encompasses a method for accessing a sub-populationof a genome, such method comprising: a) cleaving a nucleic acid samplewith a cleavage agent; b) operatively linking an adapter molecule to thecleaved ends generated in step (a), the adapter having an end compatiblewith that generated by the cleavage agent; c) mixing a primercomplementary to the adapter molecule, wherein the 3′ terminus of theprimer is complementary to the recognition sequence of the cleavageagent, under conditions that permit hybridization of the primer to theadapter; and d) adding nucleotides and a template-dependent polymerizingactivity under conditions that permit extension of the oligonucleotideprimer, the resulting population of primer extension products comprisinga sub-portion of the genome.

In a preferred embodiment, the one or more cleavage agents aresequence-specific cleavage agents.

In a preferred embodiment, the one or more cleavage agents aresequence-specific cleavage agents and the primers comprise sequencescomplementary to the recognition sequence of the sequence-specificcleavage agents.

In a preferred embodiment, the primers additionally comprise 3′ endsequences capable of hybridizing to only a subset of the molecules inthe nucleic acid sample.

It is preferred that the 3′ end sequences comprise terminal extensionsimmediately adjacent to the sequence that hybridizes to the recognitionsequence.

It is also preferred that the terminal extensions are mononucleotidesselected from the group consisting of: A, C, G, and T.

It is also preferred that the extensions are dinucleotides selected fromthe group consisting of: AA; AG; AC; AT; CA; CG; CC; CT; GA; GG; GC; GT;TA; TG; TC; and TT.

It is also preferred that the extensions are trinucleotides selectedfrom the group consisting of: AAA; AAC; AAG; AAT; AGA; AGC; AGG; AGT;ACA; ACC; ACG; ACT; ATA; ATC; ATG; ATT; CAA; CAC; CAG; CAT; CCA CCC;CCG; CCT; CGA; CGC; CGG; CGT; CTA; CTC; CTG; CTT; GAA; GAC; GAG; GAT;GCA; GCC; GCG; GCT; GGA; GGC; GGG; GGT; GTA; GTC; GTG; GTT; TAA; TAC;TAG; TAT; TCA TCC; TCG; TCT; TGA; TGC; TGG; TGT; TTA; TTC; TTG; and TTT.

In a preferred embodiment, the extensions are selected from the groupconsisting of: tetranucleotides, pentanucleotides, hexanucleotides,septanucleotides, and octanucleotides.

In a preferred embodiment, an amount of chain-terminatingdeoxynucleotide analogs is added sufficient to limit the length of theaverage extension product to between about 500 and 5000 nucleotides.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 500 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 750 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 1000 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 1500 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 2000 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 3000 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 4000 nucleotides in length.

In a preferred embodiment, an amount of chain-terminating nucleotideanalogs is added sufficient to limit the average extension product toapproximately 5000 nucleotides in length.

In a preferred embodiment, the oligonucleotide primer or one or more ofthe deoxynucleotides is detectably labeled. In a preferred embodiment,the label is selected from the group consisting of fluorescent moieties,radioactive moieties, biotin and digoxigenin.

In a preferred embodiment, the oligonucleotide primer is attached to asolid support.

In a preferred embodiment, the method for accessing a sub-population ofa genome comprises the additional step of identifying a nucleic acidsequence polymorphism in a population of individuals.

In a preferred embodiment, the method of accessing a sub-population of agenome comprises the additional step of genotyping an individual withrespect to a nucleic acid sequence polymorphism.

As used herein, the term “nucleotide sequence” refers to a consecutivelinear arrangement of nucleotide bases at least two nucleotides inlength on a nucleic acid molecule.

As used herein, the term “reference sequence” refers to a sequence inthe genome which is selected as a standard for sequence comparison. Thestandard is selected based on a sequence containing a nucleotide at oneposition in the reference sequence or nucleotides at a number ofpositions in the reference sequence which represent those nucleotidesfound most frequently at those positions in two or more individuals, orin a population or in a species. A reference sequence also may refer toa sequence selected from an individual and used for comparison to thesequence of another one or more individuals.

As used herein, the term “sequence difference” refers to one or morenucleotide differences in a given sequence with respect to a referencesequence.

As used herein, the term “nucleic acid sample” refers to a samplecontaining nucleic acid molecules including a sample comprising genomic,cDNA, mitochondrial, chloroplast, or RNA nucleic acids, a samplecomprising nucleic acids expressed by a given tissue or cell type, or asample comprising nucleic acids produced by replication of nucleic acidsexpressed by a given tissue or cell type. The term “nucleic acid sample”as used herein does not encompass synthetic random sequence DNA or RNA.

As used herein, the term “sequence-specific binding activity” refers toan activity that binds with a particular nucleic acid sequence orsequence motif. Depending upon the particular activity, asequence-specific binding activity may bind a single, invariantsequence, or it may bind two or more variant sequences with conservednucleotides at particular positions.

As used herein, the term “separation element” refers to a moiety thatcan facilitate the separation of a sub-population of nucleic acidmolecules from a larger population of nucleic acid molecules based uponrecognition of a specific sequence. A separation element according tothe invention comprises a sequence-specific binding activity which iseither immobilized or capable of being immobilized so as to effectseparation of bound nucleic acid molecules.

As used herein, the term “sequence that is bound by a sequence-specificbinding activity” refers to the particular sequence or sequence motifbound by a particular sequence-specific binding activity. As usedherein, the terms “anchor” or “anchor sequence” and “marker sequence”are equivalent to “sequence that is bound by a sequence-specific bindingactivity”.

According to the invention, such a sequence occurs at least twice, butcan occur, for example, 3, 4, 5, 10, 20, 50, 100, 1000, 10,000, 25,000,50,000 or even 100,000 times or more per genome, and can be selected foror enriched relative to regions of the genome where such sequences areabsent or present in lower abundance.

As used herein, the term “enrichment” or “enrichment of genetic markers”refers to the result of any process which increases the concentration ofany particular nucleic acid sequence (genetic marker) relative to someother nucleic acid sequence as compared to a sample not subjected to theprocess. As used herein, a nucleic acid sample is considered to beenriched for a particular marker if the marker is in greaterconcentration relative to the average concentration of all markers thanin a sample which has not been subjected to an enrichment process; forexample, where the marker is present in the enriched sample at aconcentration 5-fold greater than in the unenriched sample. As usedherein, the “complexity” of a DNA sample refers to the number ofdifferent unique sequences present in that sample.

As used herein, a sample is considered to have “reduced complexity” ifit is less complex (for example, in the range of 5-fold to 10-fold,inclusive, less complex) than the DNA sample from which it is derived.

As used herein, “solid support” refers to a solid or semisolid materialwhich has the property, either inherently or through attachment of somecomponent conferring the property (e.g., an antibody, streptavidin,nucleic acid, or other affinity partner), of binding to a nucleic acidor polypeptide. Such binding can be direct, or may be mediated by alabel (e.g., biotin, a nucleic acid sequence tag, or other affinitypartner) attached to the nucleic acid or polypeptide. Examples of solidsupports include, but are not limited to nitrocellulose and nylonmembranes, agarose or cellulose based beads (e.g., Sepharose) andparamagnetic beads.

As referred to herein, the term “cleavage agent” refers to an agent ormolecule having an activity that cuts a nucleic acid molecule. It shouldbe understood that a cleavage agent as used herein may cleave one orboth strands of a double-stranded nucleic acid molecule.

As used herein, the term “sequence-specific cleavage agent” refers to acleavage agent that requires or recognizes the presence of a particularnucleic acid sequence. or “recognition sequence” for cleavage to occur.A sequence-specific cleavage agent may cleave the nucleic acid eitherwithin the recognition sequence or at a point removed from therecognition sequence on the same molecule.

As used herein, the term “subset of fragments” or “subset of molecules”refers to that fraction of a population of nucleic acid fragments, lessthan every molecule in the population, having a given characteristic(e.g., having ends capable of annealing to a particular linker orprimer, or having a particular average length).

As used herein, the term “sequences capable of being operatively linked”refers to nucleic acid sequences that can be annealed to anotherparticular nucleic acid sequence by Watson:Crick hydrogen bonded basepairing, or annealed and ligated. However, covalent attachment is notalways necessary for there to be an operative linkage.

As used herein, the term “molecule capable of being replicated” refersto a nucleic acid molecule that permits the synthesis or polymerizationof copies or replicas of itself or a nucleic acid molecules linked toit. As used in this context, a complementary strand of a nucleic acidfalls within the meaning of the term “replica”. The term “moleculecapable of being replicated” includes, but is not limited to, anoligonucleotide or a plasmid.

As used herein, the term “system capable of replicating said linkedmolecules” refers to the components (such as oligonucleotide primers anda template-dependent nucleic acid polymerizing activity, or host cells)necessary for in vitro or in vivo generation of a copy or replica of amolecule either annealed or linked to a molecule capable of beingreplicated.

As used herein, the term “library” refers to a collection of nucleicacid sequences linked to nucleic acid molecules that permit thereplication of the members of the collection within host cells.

As used herein, the term “hybridize specifically” means that nucleicacids hybridize with a nucleic acid of complementary sequence. As usedherein, a portion of a nucleic acid molecule may hybridize specificallywith a complementary sequence on another nucleic acid molecule. That is,the entire length of a nucleic acid sequence does not necessarily needto hybridize for a portion of such a sequence to be considered“specifically hybridized” to another molecule; there may be, forexample, a stretch of nucleotides at the 5′-end of a molecule that donot hybridize while a stretch at the 3′ end of the same molecule isspecifically hybridized to another molecule.

As used herein, the term “infrequently,” as applied to cleavage ofmammalian DNA (e.g. human DNA) by a restriction endonuclease refers tocleavage which occurs 300,000 times or less in a given genome (forexample, 250,000, 200,000, 150,000, or 100,000 times) or which generatesan average fragment size of 10,000 bp or more (for example, 20,000 bp,30,000 bp, 50,000 bp) when a given genomic DNA sample is digested. Thesefrequencies are particularly applicable to human DNA. Restrictionendonucleases that generate average fragment sizes of 10,000 base pairsor more on human DNA include, but are not limited to AscI, BssHII, EagI,NheI, NotI, PacI, PmeI, RsrII, SalI. SbfI, SfiI, SgrAI, SpeI, SrfI, andSwaI.

As used herein, the term “frequently,” or “more frequently” as appliedto cleavage of mammalian DNA by a restriction endonuclease refers tocleavage which occurs more than 300,000 times in a given genome (forexample, 500,000-1,000,000 times) or which generates an average fragmentsize smaller than 10,000 bp (for example, 2,000 bp, 5,000 bp, 8,000 bp)when a given genomic DNA sample is digested. These frequencies areparticularly preferred for human DNA.

As used herein, the term “average fragment length” refers to a length ofnucleic acid molecules in a particular population of nucleic acidmolecules which generally is approximately (i.e., within 50-150% of) apredetermined length. In cases where restriction endonucleases are usedto generate fragments of a chosen average fragment length, it should benoted that while the frequency of cutting for a particular sequence maybe generally predicted based on the length of the recognition sequence,the base composition of the recognition sequence, and the size orsequence content of the genome, the fragment sizes for a givenrestriction enzyme may not fall on a bell-shaped curve. In fact, theremay be a bimodal or multi-modal distribution. For example, therestriction enzyme recognition sequence may happen to occur in asequence that is highly repeated in the genome. Such an occurrence willcause there to be a “shoulder” in the normal distribution of fragmentlengths. Similarly, if the recognition sequence occurs in two differentrepeated elements, there will be two “shoulders” in the distribution,etc. In practice, the average size of fragments generated by a givenrestriction endonuclease may be estimated by examination of fragmentsafter electrophoretic separation on a gel. One should recognize,however, that larger fragments stain more intensely than do shorterfragments on such a gel.

As used herein, the term “genotyping an individual with respect to anucleic acid sequence polymorphism” refers to the identification of thenucleic acid sequence of an individual at a site known to have one ormore polymorphisms in a population of other individuals. Within thiscontext a “population of other individuals” can be one or more otherindividuals.

As used herein, the terms “sub-population of a genome” or “sub-portionof a genome” refer to a collection of nucleic acids derived from agenomic nucleic acid sample wherein the collection does notsubstantially contain sequences representative of the entire genome.

As used herein, the term “incomplete extension product” refers to thenucleic acid products of primers or promoters extended by atemplate-dependent nucleic acid polymerizing activity in whichpolymerization proceeded over less than the full length of the templatemolecule, or in the case where there is a primer binding site orpromoter on both ends of each template, then less than one half of thelength of the template molecule. An incomplete extension product may be10 nt, 20 nt, 100 nt to 5000 nt or more in length, e.g., 100 nt to 1000nt, 200 nt to 800 nt, 400 nt to 700 nt, or 500 to 600 nt.

The inventive methods provide significant improvements over prior artmethods for identifying nucleotide sequence differences which arecurrently laborious, relatively expensive, and time consuming.Genotyping studies useful for pharmacogenomics studies, for example, mayinvolve 100,000 or more polymorphic markers per study subject. Theinventive methods provide simplification of the processes for obtainingsuch markers and decrease the cost of large-scale genotyping efforts.The invention thus provides for identification of polymorphic markers,but also is applicable to any type of genetic marker, such as (withoutlimitation) tandem repeat sequences, deletions and insertions.

DETAILED DESCRIPTION

The present invention recognizes that a significant problem encounteredin the identification of nucleic acid sequence polymorphisms relates tothe complexity of the genome. The invention is predicated upon theobservation that a nucleic acid marker sequence bound by asequence-specific binding activity may be used to facilitate theidentification of polymorphisms.

The human genome is complex. There are approximately 3 billionnucleotides per haploid human genome. A single polymorphic nucleotidemust be identified in the presence of 3 billion other nucleotides,requiring an assay with extreme sensitivity and specificity. Theinvention provides methods that reduce the complexity of the genome orenrich for a particular subset of sequences that will facilitate theidentification of sequence polymorphisms.

The invention disclosed herein recognizes that any nucleic acid sequencebound or recognized by a sequence-specific binding activity may be usedto reduce the complexity of the genome to facilitate the identificationof polymorphisms. The methods disclosed herein solve the genomiccomplexity problem by identifying and utilizing a marker sequence thatcan be enriched for with a simple anchored enrichment procedure.Molecules comprising the marker sequence represent a sub-population ofthe genome or nucleic acid sample having reduced complexity.

Polymorphisms, particularly single nucleotide polymorphisms (“SNPs”),are essentially randomly distributed throughout the genome. The use ofthe methods of the invention, through the enrichment for moleculesbearing a marker sequence, allows substantially reproducible access tosubstantially similar reduced-complexity sub-populations in differentindividuals in a population or even in different samples from a singleindividual. Because polymorphisms are essentially randomly distributedthroughout the genome, a number of polymorphic sequences will be presentin the reduced-complexity population of nucleic acid molecules bearing agiven marker sequence. Such reduced-complexity subpopulations may thenbe analyzed to either identify, polymorphisms or to determine thegenotype of polymorphic loci within that sub-population.

A significant advantage of the methods of the invention is that theypermit accession of a substantially similar reduced-complexitysub-population of nucleic acid molecules from any individual in a givenspecies. The reduced-complexity sub-population of nucleic acids may thenbe genotyped with regard to polymorphisms in the sub-population ofnucleic acids using any of a number of methods known in the art. Thereduced complexity of the nucleic acid population used for genotypeanalysis allows for an increased signal to background ratio in thegenotyping methods.

For example, a DNA molecule carrying a sequence that can be recognizedby a sequence-specific binding molecule, such as the DNA binding proteinGal4, can be separated from DNA not carrying the Gal4 sequence. If suchDNA is exposed to Gal4 protein which is bound to a solid support, theDNA molecules carrying the Gal4 binding sequence can be separated fromother DNA molecules by washing of the Gal4:DNA complexes to removeunbound DNA. If target genomic DNA is first sheared intosub-genome-sized fragments of a desired size and then subjected to aGal4 protein separation step, any polymorphic markers contained on theGal4-bound DNA fragments will be enriched relative to markers on DNAfragments not bound by Gal4 protein. The enriched sub-fraction of thegenome (sub-genome) may then be tested for the presence or absence ofparticular polymorphic alleles through various assays, such asallele-specific hybridization (Saiki, 1986, supra), primer extension(Pastinen et al., 1997, Genome Res. 7: 606), the oligonucleotideligation assay (Nickerson et al., 1990, Proc. Natl. Acad. Sci. USA 87:8923), or the Invader™ assay (Third Wave Technologies; Rosetti et al.,1997, Mol. Cell. Probes 1:155), among others (see below).

Any molecule that binds to a recognition sequence in a nucleic acid canbe used to enrich for molecules bearing a marker sequence. Thus, anysequence bound by a sequence-specific binding activity may be used as amarker according to the invention. For example, DNA binding domains suchas found in transcription factors (Jun, Fos, etc.), proteins withzinc-finger DNA binding regions, restriction endonuclease recognitiondomains, sequence-specific antibodies, nucleic acid molecules, aptamers,peptide nucleic acid (PNA) molecules, peptides and affinity resins thatrecognize DNA having particular GC content or methylation status may allbe used according to the invention.

Marker sequences facilitate the identification of polymorphic sequences.Any sequence variation between a) two individuals, or between b) anindividual and a population of individuals or between c) twopopulations, or between d) one or more individuals and a species as awhole may represent a polymorphism. When compared to sequences withinthe general population, a polymorphism is typically present at afrequency of about 1% or greater, however the term can apply to anysequence variation between two or more individuals in a population,regardless of the frequency. For example, a polymorphism may be presentat a frequency of 0.001% (that is, present in at least one individualper 100,000 individuals), 0.01%, 0.1%, 1% or even 10% or more in a givenpopulation of individuals.

A polymorphism may be an insertion, deletion, duplication, orrearrangement of any length of a sequence, including single nucleotidedeletions, insertions, or base changes (herein referred to as “singlenucleotide polymorphisms” or “SNPs”). A polymorphism, including a SNP,may be neutral or may have an associated variant phenotype. A “neutralpolymorphism” is a polymorphism wherein a phenotypic change has not beenfound in individuals with the sequence variation. A “functionalpolymorphism” is a sequence variation that has an associated alteredphenotype, and typically occurs at a frequency of greater than or equalto 1% in the population. The term “mutation” generally refers to agenetic change that occurs at a frequency of less than or equal to about1% in a population, and may, but not necessarily, be associated with aphenotypic change.

The inventive methods, i.e., of discovering polymorphisms are useful,for example, in the field of pharmacogenomics, which seeks to correlatethe knowledge of specific alleles of polymorphic loci with the way inwhich individuals in a population respond to particular drugs.

A broad estimate is that for every drug, between 10% and 40% ofindividuals do not respond optimally. Several well known examples(particularly the association of the response or lack of response to theAlzheimer's drug Tacrine with one's genotype at the ApoE locus (Farlowet al., 1998, Neurology, 50: 669) suggest that allelic differences thataffect drug absorption, retention, general metabolism and clearance maybe involved in these observed differences.

In order to create a response profile for a given drug, the genotypewith regard to polymorphic loci of those individuals receiving the drugmust be correlated with the therapeutic outcome of the drug. This isbest performed with analysis of a large number of polymorphic loci. Oncea genetic drug response profile has been established by analysis ofpolymorphic loci in a population, a clinical patient's genotype withrespect to those loci related to responses to particular drugs must bedetermined. Therefore, the ability to identify the sequence of a largenumber of polymorphic loci in a large number of individuals is criticalfor both establishment of a drug response profile and for identificationof an individual's genotype for clinical applications.

Single nucleotide polymorphisms are, by far, the most prevalent form ofgenetic polymorphism, and as such, they are useful to correlate drugresponses with profiles of individual genetic variation to predictpatient responses to drugs. The polymorphisms need not necessarily be ingenes related to the particular disease being treated with a given drug.Rather, in addition to polymorphisms occurring in disease-related genes,useful polymorphisms for establishing drug response profiles can occurin genes or genetic control elements (enhancers, promoters, processingsignals and the like) which ultimately have an effect at any step in themetabolism and clearance of the drug or its metabolites. For thatmatter, useful polymorphisms may simply be closely genetically linked toa gene or control element involved in a drug response, without actuallybeing a part of the coding or regulatory sequences.

Reduction of the Complexity of Nucleic Acid Samples Using MarkerSequences

In one embodiment, the method of the invention incorporates the use of acomplexity reduction mechanism to both produce the DNA to be used tofind polymorphic markers and to reduce the complexity of the DNAtemplate prior to genotyping. Thus, polymorphic sequences are discoveredusing the marker sequence to enrich a subset of the genome, and the sameenrichment mechanism is used to reduce the complexity of the genomeprior to genotyping. In the following discussion, for clarity, the useof sequence-specific binding molecules according to the invention isdescribed. The sequence specific binding molecules are a pair ofcleavage agents, the restriction enzymes NotI and EcoRI, which arerepresentative of a combination of two cleavage agents wherein a firstagent cleaves genomic DNA infrequently and a second agent cleaves morefrequently than the first.

In a preferred embodiment of the method of the invention, therestriction enzymes NotI and EcoRI are first used to cleave humangenomic DNA according to standard methods. The doubly cleaved fragmentsare then ligated into a NotI/EcoRI cloning vector to produce asub-library of the human genome. The sub-library consists substantiallyof the DNA flanking most NotI sites. There are approximately 30,000 NotIsites in the human genome. Thus, a library with 1× representation shouldcontain about 60,000 clones. To discover common polymorphisms in thissubset of the genome, one would then sequence 60,000 or more clones fromlibraries constructed from at least one, and preferably severalindividuals. The DNA sequencing is performed using vector-specificprimers, entering the human DNA from the NotI end and the EcoRI end. IfDNA from four individuals is used, for example, one would sequenceapproximately 60,000×2×4=480,000 segments to achieve a 4× representationaround the NotI sites represented in the library. With 4× representationof the NotI/EcoRI sub-genome, one would expect to sequence sample eachNotI/EcoRI fragment at least twice for 90.8% of such fragments (1 minusPoisson (0 or 1 with a mean of 4)). One would expect to sample about 57%of the fragments 4 or more times (1 minus Poisson (0, 1, 2, or 3 with amean of 4)). Alternatively, one may also make libraries containingNotI/NotI fragments, and sequence both ends of these. Because the NotIrecognition sequence is an 8 bp sequence composed entirely of C and G,such fragments are likely to comprise CpG islands found near transcribedregions of the genome. Thus, the subset of the genome represented bylibraries made using NotI cleavage will likely be biased towardstranscribed regions. An advantage of this particular method, therefore,is that polymorphisms identified from libraries made using NotI willlikely fall near or within protein-encoding sequences.

Methods of the invention that utilize library construction or cloning ofenriched sequences to replicate enriched populations require theselection of appropriate vector and host combinations. Vectors and hostssuitable for libraries or other cloning according to the methods of theinvention are well known in the art, however preferred attributes of avector and host for use in the methods of the invention are discussedbelow.

A sequence selected or enriched according to the methods of theinvention may be inserted into a vector in a forward or reverseorientation. A vector may include regulatory sequences, including, forexample, a promoter, operably linked to the sequence. A vector may alsocontain a gene to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli. The vector may also include an originof replication to ensure maintenance of the vector and, if desirable, toprovide amplification within the host.

The vector containing the DNA sequence enriched as described herein, aswell as an appropriate promoter or control sequence, may be employed totransform an appropriate host. Many suitable vectors and promoters areknown to those of skill in the art, and are commercially available. Thefollowing vectors are provided by way of example. Bacterial: pQE70,pQE60, pQE-9 (Qiagen), pBS, phagescript, pBluescript SK, pBSKS,LambdaZAP, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A,pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo,pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL(Pharmacia). However, any other plasmids or other vectors may be used aslong as they are replicable and viable in the host.

Promoter regions can be selected from any characterized gene andincorporated into appropriate vectors using techniques well known in theart. Bacterial promoters useful according to the invention include, butare not limited to lacI, lacZ, T3, T7, gpt, λP_(R), λP_(L) and trp.Eukaryotic promoters include, but are not limited to CMV immediateearly, HSV thymidine kinase, early and late SV 40, L TRs fromretrovirus, and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart.

A host cell may be a higher eukaryotic cell, such as a mammalian cell,or a lower eukaryotic cell, such as a yeast cell, or the host cell maybe a prokaryotic cell, such as a bacterial cell. Examples of appropriatehosts include but are not limited to: bacterial cells, such as E. coli,Bacillus subtilis, Salmonella typhimurium and various species within thegenera Pseudomonas, Streptomyces, and Staphylococcus, although othersmay also be employed as a matter of choice; fungal cells, such as yeast;insect cells such as Drosophila and Sf9; animal cells such as CHO, COSor Bowes melanoma; plant cells, etc. The selection of an appropriatehost is within the scope of one of skill in the art from the teachingsherein.

Introduction of the construct into the host cell can be effected bycalcium transfection, DEAE-Dextran-mediated transfection, liposomemediated transfection, or electroporation (Ausubel et al., 1992, ShortProtocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc.,pp. 9-5 to 9-14) in the case of eukaryotic cells.

Prokaryotic cells may be made competent to take up foreign DNA bystandard methods (Ausubel et al., supra, 1992, pp. 1-22 to 1-23) knownin the art. Recombinant constructs may be introduced to bacteria bystandard transformation (for plasmids) or transfection/infection (forphage DNA or phage particles; see Ausubel et al., supra, 1992, pp. 1-22to 1-23).

Nucleic Acid Complexity Reduction Methods Useful in the Invention:

The invention contemplates in part the use of complexity reduction andsequence enrichment methods. Complexity reduction reduces the number ofunique sequences present in a nucleic acid sample, and enrichmentincreases the relative concentration of a particular sequence or subsetof sequences in a nucleic acid sample.

A subset of nucleic acid molecules, each containing a sequence bound bya sequence-specific binding activity, is prepared as follows accordingto the invention. Although cleavage agents are used in the followingmethod, other sequence-specific binding activities maybe used accordingto the invention.

The concentration of molecules bearing marker sequences in a populationof nucleic acid molecules can be enriched by cleaving genomic DNA withone or more restriction enzymes, and then enriching for a sub-populationof the DNA fragments. Genomic DNA can be cleaved with a restrictionenzyme, for example SalI, which cleaves somewhat infrequently in genomicDNA. The DNA may then be cleaved with a second restriction enzyme thatcleaves more frequently (for example, EcoRI). The doubly-cleaved DNA canthen be cloned into a replication vector specific for DNA cleaved byboth enzymes (e.g. a SalI/EcoRI cloning vector). Such cloned DNA willhave been enriched for sequences surrounding SalI sites relative to theDNA that was not cloned into the vector.

Alternatively, the SalI ends can be ligated to adapter molecules thatspecifically hybridize with the SalI sequence overhangs. The DNA maythen be sheared, or cleaved with a second restriction enzyme, such asEcoRI. If the adapter molecules are attached to a solid support, orcarry a biotin moiety or some other moiety capable of being attached toa solid support, the SalI-terminated fragments may then be separatedfrom the other fragments in the mixture and thus enriched inconcentration. Polymorphic markers contained on fragments in suchenriched populations of nucleic acid molecules may then be assayed withmethods described herein below.

A marker sequence may also be enriched for in a population of nucleicacid molecules by cleaving the nucleic acid (preferably, but notnecessarily with a restriction endonuclease; any sequence-specificcleavage agent is acceptable for enrichment according to this method)and then binding (or, alternatively, binding, then cleaving) moleculesbearing a marker sequence to a marker sequence-binding activity.Conditions for specific binding, as well as washing conditions forremoval of non-specifically bound DNA molecules will necessarily varydepending upon the nature of the DNA binding activity employed and maybe adjusted by one of skill in the art. Generally, it is clear that tobe selected for this type of use, the sequence specific binding activityand the conditions under which such activity functions will be wellknown prior to its selection for this purpose.

Another method of enriching for molecules with a particular markersequence involves the use of non-specific PCR or repeat-sequence PCR.Inter-Alu PCR has been used to amplify a subset of the human genome(Nelson et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6686; Sadhu etal., 1992, Genomics 14: 728). Primers designed to amplify other repeatedsequences, such as SINEs and LINEs have also been made (Cotter et al.,1990, Genomics 7:257: Ledbetter et al., 1991, Genomics 8: 475-481).Primers that identify such repeated sequences in the genome eitheramplify all DNA between two points in each amplifiable repeat sequence,or amplify DNA between different adjacent copies of a repeated sequence.Such complexity reduction and enrichment might allow one to detectmarkers without prior template amplification, however highly repeatedsequences such as Alus, SINES, and LINEs are difficult to differentiatefrom each other. A marker set contained within such repeat elementswould be difficult to locate on a physical map. Moreover, certainmarkers would be located in copies of the repeat element that areidentical to other copies in the genome (at least in the region beingtested for the presence of the marker). However, markers located betweenrepeat elements would be useful. Primers that would amplify thesequences between repeat elements could be used to enrich a populationof nucleic acid molecules for sequences containing useful markers.

DNA amplification using arbitrary PCR primers produces DNA fragmentsthat are mostly unique in sequence (Welsh & McClelland, 1990, Nucl.Acids Res. 18:7213; Williams et al. 1990, Nucl. Acids Res. 18:6531). Twopublished methods are respectively called AP-PCR for arbitrarily primedPCR and RAPD for random amplified polymorphic DNA. AP-PCR generally useslonger PCR primers than RAPD. In both methods, an arbitraryoligonucleotide primer is selected and used to amplify genomic DNA underrelatively non-stringent conditions. The primers are extended atmultiple locations around the genome, yet yield exponentialamplification only in those regions where the primers have hybridized ashort distance from another hybridized primer and where both primers arehybridized to opposite strands of the genomic DNA. Multiple arbitraryPCR fragments are produced. The particular fragments are reasonablyreproducible from experiment to experiment, provided that similaramplification conditions are used (Schweder et al., 1995, Biotechniques19:38; Ellsworth et al., 1993, Biotechniques 14: 215). The lengths ofsuch fragments have been analyzed by electrophoresis and used as markersfor the presence or absence of particular DNA sequences in the genomictemplate. PCR fragment differences between individuals or between twochromosomes in a single individual result from polymorphic differencesin the template regions to which the arbitrary primers bind. Methodsinvolving the simultaneous use of two arbitrary primers have also beenreported to yield consistent fragment patterns (Hu et al., 1995, PCRMethods and Applications 4:346; Desmarais et al., 1998, Nucl. Acids Res.26:1458).

AP-PCR or RAPD may be used to reduce the complexity of the genome foridentification of polymorph isms or for genotyping individuals withrespect to the polymorphisms. The major DNA bands seen in a gelelectrophoresis of AP-PCR or RAPD products are thought to representabout 15 kilobases of the genome (Desmarais et al., 1998, Nucl. AcidsRes. 26: 1458). This is an enormous reduction of the complexity of thehuman genome, with sequences present in 15 kilobases roughly equivalentto about 1/1000 of the yeast genome, and 1/200,000 of the human genome.One may thus pool 1000 AP-PCR reactions; the complexity of the resultingproduct would be about equivalent to the yeast genome. In addition, onemay perform less stringent AP-PCR reactions or multiplex the AP-PCRreactions, thereby increasing the percentage of the genome beingamplified in one reaction. Fragments most abundant in the mix would bethe ones amplified most often. Thus, polymorphisms discovered in thisreduced-complexity population tend to be those present on the mostabundant fragments in the AP-PCR product.

Ideally, AP-PCR conditions may be adjusted (by varying arbitrary primerlength, number of primers, and/or annealing conditions) to amplify about1/200^(th) of the human genome in one tube. This product is about ascomplex as the yeast genome and may be hybridized directly toallele-specific probes using the methods of Winzeler et al., which candetect polymorphisms in the yeast genome without enrichment (Winzeler etal., 1993. Science 281: 1194). If common polymorphisms occur with aheterozygosity of approximately 1 in 1500 bases, then approximately10,000 polymorphisms should be present in such a 1/200^(th) sub genomefrom a single individual. Thus, it is possible to genotype 100,000polymorphisms using about 10 AP-PCR reactions where approximately1/200^(th) of the genome is being amplified per AP-PCR reaction. Eachreaction is then hybridized to an array capable of detecting 10,000different polymorphisms using the methods of Winzeler et al. Thisgreatly decreases the time and expense required to obtain genotypic datafrom 100,000 polymorphic markers per individual. The genotype of anindividual with respect to the polymorphic markers found in theNotI/EcoRI (or other similarly constructed) sub-libraries as describedabove can be determined using the Nod recognition sequence as a markerto generate a reduced-complexity portion of the genome containing knownpolymorphisms. One way to accomplish this is to cleave genomic DNA withNotI, ligate it to a NotI specific adapter, cleave the DNA with EcoRI,attach the adapter-Nod/RI fragment complexes to a solid support (e.g.,via the adapter), wash away unattached DNA, cleave the DNA with NotI andelute the reduced complexity sample. Such a sample will be about 20 foldless complex than the entire genome. If the entire genome contains 3billion nucleotides, and there are 30,000 NotI sites, and the averageNotI/RI fragment is about 2,500 nucleotides in length, then the NotI/RIfragments will represent about 30,000×2×2500/(3×10⁹)=5% of the genome.Assays used for the detection of these anchored polymorphisms will thushave a 20 fold higher signal to noise ratio than assays used to detectpolymorphisms in un-enriched genomic DNA.

Likewise, other methods may be used to reduce the complexity of thegenome to a higher degree, proportionately increasing the signal tonoise ratio. As noted above, the yeast genome is about 200 fold lesscomplex than the human genome, and it is possible to detect singlenucleotide changes in the yeast genome without complexity reduction(Winzeler et al., 1993, supra). Therefore, it is possible to use theNotI/RI complexity-reduction strategy, coupled with a 10 fold linearamplification strategy to produce template DNA of sufficiently lowcomplexity for direct polymorphism detection.

Linear amplification can be carried out in several ways, either coupledwith a physical enrichment strategy or performed independently. Onemethod utilizes primers that recognize the NotI adapters (oranchor-specific adapters) that have been ligated to the genomic DNA. Ifsuch primers are designed so that they recognize the NotI adaptersequence and, in one embodiment, also recognize the partial Not sequencethat is ligated to the adapter, they can prime DNA synthesis (by any ofa number of enzymes known to those skilled in the art) starting at theNotI ends of the genomic DNA. The complexity of the newly synthesizedDNA can be further reduced by including a 3′-terminal extension on theprimer immediately adjacent to the NotI recognition sequence. Dependingupon the length of the extension, different sized subsets of thepopulation will be capable of extension, and thereby enrichment withconcomitant reduction in complexity.

For example, a 3′-mononucleotide extension of G, A, T or C immediatelyadjacent to the sequence complementary to the marker sequence will allowthe extension of roughly one quarter of the sequences bearing the markersequence, for an approximate 4-fold further reduction in complexity.Similarly, a 3′-terminal dinucleotide extension in any of the 16possible combinations (AA, AC, AG, AT, CA, CC, CG, CT, GA, GC, GG, GT,TA, TC, TG, and TT) will allow extension of roughly 1/16^(th) of thesequences bearing the marker sequence, for an approximately 16-foldfurther reduction in complexity. By the same reasoning, a 3′-terminaltrinucleotide extension in any of the 64 possible combinations (AAA,AAC, AAG, AAT, AGA, AGC, AGG, AGT, ACA, ACC, ACG, ACT, ATA, ATC, ATG,ATT, CAA, CAC, CAG, CAT, CCA, CCC, CCG, CCT, CGA, CGC, GGG, CGT, CTA,CTC, CTG, CTT, GAA, GAC, GAG, GAT, GCA, GCC, GCG, OCT, GGA, GGC, GGG,GGT, GTA, GTC, GTG, GTT, TAA, TAC, TAG, TAT, TCA, TCC, TCG, TCT, TGA,TGC, TGG, TGT, TTA, TTC, TTG and TTT) will effect an approximately64-fold reduction in complexity. Further reductions may be achievedusing 3′-terminal extensions of 4, 5, 6, 7, 8 or more nucleotidesimmediately adjacent to the sequence complementary to the markersequence. Using this complexity reduction scheme, one may analyze theentire population, if necessary, through use of primers bearing all 4,16, 64, 256, etc., possible 3′ extensions.

Primer extension products made using, for example, dinucleotide-extendedprimers, are, in theory, 16 fold less complex than the 5% of the genomeselected by the NotI/EcoRI method. Not all of the 16 primers will primewith equal efficiency, and some of the primers will be somewhatpromiscuous in their priming (for example when G in the primer ortemplate is opposing T in the template or primer there will be someextension of the primer even though a mismatch occurs between one of the3′ nucleotides of the primer and the template). Overall, however, theprimer extension products will be less complex than the NotI/RIsubgenome. In this particular case, as well as in cases like it, theprimer extension product complexity will be approximately similar to thecomplexity of the yeast genome, such that the methods of Winzeler, etal. (1993, supra) can be used to detect polymorphisms in the primerextension products. Alternatively, such DNA synthesized from the 16different primers (used separately) can be used as a template for otherpolymorphism genotyping methods as referenced herein (see below).

For example, genomic DNA may be cleaved with NotI (or anotherinfrequently cleaving cleavage agent), ligated with a NotI (or othersequence) specific adapter, and then either used directly for primerextension or further purified. Further purification can be achieved bycleaving the DNA with EcoRI (or other cleavage agent) followed byisolation of the NotI/EcoRI and NotI/NotI fragments using a captureelement attached to the NotI adapters (non-limiting examples include abiotin moiety on the adapter and streptavidin on a solid support or adigoxigenin moiety on the adapter and an anti-digoxigenin antibody on asolid support; conditions for capture, as well as methods for biotin ordigoxigenin labeling of oligonucleotides are well known to those skilledin the art). The purified subgenome is then mixed with one of 16different primers bearing 3′-terminal dinucleotide extensions underconditions permitting primer annealing. These primers will anneal to theNotI adapter sequence and the partial NotI recognition sequence ligatedto the adapter. However, only in the approximately 1 out of 16 linkedmolecules bearing the complement of the 3′-terminal dinucleotideextension adjacent to the NotI sequence will an extension product begenerated.

The primer-template complexes are then placed into reaction conditionscapable of synthesizing DNA (e.g. dNTPs, polymerase, buffer; one set ofappropriate conditions is as described herein for PCR, with optimalannealing temperature determined using the formulae also describedherein below) and the primers extended from the variable dinucleotideends to the opposite ends of the template. The primer extension productsmay be labeled by including one or more labeled dNTPs in the extensionreaction. Labels include, but are not limited to radioactive orfluorescent moieties, biotin and digoxigenin. A cycling reaction can beused to linearly amplify the amount of primer extension products, ifnecessary, for subsequent assays. Labeled primer extension products maythen be analyzed for the genotype with respect to particularpolymorphisms, for example, allele-specific hybridization as describedherein.

It should be clear to one skilled in the art that purification usingsome solid phase affinity separation, as outlined above, is notnecessary. The DNA can be digested with a cleavage agent, such as NotI,and ligated with specific adapters. Primers specific for the adapters,the NotI site, and in some cases several nucleotides of the genomicsequence adjacent to the Not site, can be used to create primerextension products that will be enriched for the regions adjacent to theNotI site. The lengths of such primer extension products can becontrolled by adding chain terminating nucleotides to the primerextension mix. The ratio of chain terminating nucleotide triphosphatesto normal nucleotide triphosphates will influence the average length ofprimer extension products. Such primer extension products may either belabeled or unlabeled.

These methods allow the complexity of genomic DNA to be significantlyreduced simply through cleavage, annealing and/or ligation, DNA capture(optionally), and a small number (in the example cited, 1 to 16) ofprimer extension reactions. The methods provide subpopulations of thegenome that can be directly analyzed for the presence of polymorphismswith lower background and higher efficiency than methods that do notreduce the complexity of the genomic DNA.

Methods of Genotyping Useful in the Invention:

There are a number of methods known in the art that are capable ofdetecting single nucleotide sequence differences with respect to areference sequence. Several of these are described below, however itshould be understood that any method that allows the determination ofthe sequence of a particular individual at a particular site may be usedto detect sequence differences with respect to a reference sequence inthe reduced-complexity nucleic acid populations generated according tothe invention. Direct DNA sequencing according to the classical Sanger(dideoxynucleotide sequencing; Sanger et al., 1975, J. Mol. Biol.,94:441) or Maxam & Gilbert (chemical sequencing; Maxam et al., Proc.Natl. Acad. Sci. U.S.A., 1977, 74:560) methods is capable of detectingnucleotide differences according to the invention. In the Sanger method,a primer that hybridizes to a known sequence on the molecule is extendedin the presence of a limiting amount of a chain-terminating nucleosideanalog such that a ladder of extension products of different lengths allending with that nucleotide is generated. Reactions using the sameprimer with chain-terminating analogs of each of the four dNTPsindividually allows determination of the DNA sequence followingelectrophoresis of the four reactions alongside each other on the samedenaturing gel.

Direct DNA sequencing generally requires amplification of the targetsequence. However, marker-based nucleic acid sequence enrichment methodsas described herein can raise the template concentration to levels wheredirect sequencing may be effective in determining a single nucleotidesequence difference.

DNA sequencing as necessary for certain embodiments of the invention mayalso be performed with the Exonuclease Resistance method (Mundy, U.S.Pat. No. 4,656,127), primer-guided microsequencing (Kohmer et al., 1989,Nucl. Acids Res., 17:7779), minisequencing (Pastinen et al, 1997, GenomeRes., 7: 606), extension in solution using ddNTPs (Cohen et al., FrenchPatent No. 2,650,840; PCT Application No. WO91/02087), Genetic BitAnalysis™ (GBA; Goelet et al., PCT Application No. 92/15712),ligase-polymerase-mediated GBA (Nifikorov et al., U.S. Pat. No.5,679,524) and oligonucleotide ligation assay (OLA; Landegren et al.,1988, Science 241: 1077) methods as described below.

The exonuclease resistance method (Mundy, supra) involves the use of aprimer complementary to the allelic sequence immediately 3′ to thepolymorphic nucleotide and an exonuclease-resistant nucleotidederivative. The primer is allowed to hybridize to a target moleculecontained in a DNA sample obtained from an individual, followed byaddition of the exonuclease-resistant nucleotide derivative and apolymerase. If the polymorphic site on the target DNA contains anucleotide that is complementary to the particular exonuclease-resistantnucleotide derivative, then that derivative will be incorporated intothe primer by the polymerase, rendering the primer resistant to nucleasedigestion. Because the identity of the nucleotide derivative is known,this method can unambiguously identify the nucleotide present at thepolymorphic site.

Microsequencing methods, for example as described by Kohmer et al.(Kohmer et al., supra), involve reactions containing a single labeleddeoxynucleotide as the only deoxynucleotide present in the reaction, anda primer complementary to the allelic sequence immediately 3′ of thepolymorphic site. If the primer becomes labeled upon addition of apolymerase and the labeled deoxynucleotide, the nucleotide present atthe polymorphic site must be complementary to that deoxynucleotide.

A variation on the micro sequencing method was described by Pastinen andcoworkers (Pastinen et al., supra). Briefly, the primers are designed sothat their 3′ ends hybridize immediately adjacent to each suspectedpolymorphic site, such primers would comprise sequence specific tags foreach polymorphic locus. The primers are then extended with DNApolymerase in the presence of 4 different dideoxynucleosidetriphosphates. Each dideoxynucleoside triphosphate is labeled with adifferent fluorescent molecule. The polymerase is only able to add onenucleotide to each primer, and this identifies the nucleotide in thetemplate immediately adjacent to the 3′ end of the primer, and thus thegenotype with respect to the polymorphism. The polymerase reactions canbe cycled, such as by thermocycling, to increase the amount of product.After the polymerase extension reactions, the primers are thenhybridized to a capture array bearing the various sequences containingthe polymorphisms. The spots on the capture array will producefluorescent signal if the primers were extended with a fluorescentlylabeled dideoxynucleoside triphosphate. The colors emitted from thespots reveal the alleles present in the target nucleic acid sample. Thesize of the capture array and the number of primers can be increased asneeded.

The method using extension in solution and ddNTPs, as described by Cohenet al. (supra) also involves a primer that is complementary to sequencesimmediately 3′ to a polymorphic site. The method determines the identityof the nucleotide of that site using labeled dideoxynucleotidederivatives, which, if complementary to the nucleotide at thepolymorphic site will become incorporated onto the terminus of thehybridized primer. Genetic Bit Analysis™, or GBA™ is described by Goeletet al. (supra). This method is similar to the method of Cohen et al.,except that it is preferably a heterogeneous phase assay, in which theprimer or the target molecule is immobilized to a solid phase. It isthus easier to perform, more accurate, and better suited for highthroughput analyses than the Cohen et al. method.

The Oligonucleotide Ligation Assay or OLA was described by Landegren etal. (supra). This is also a solid phase assay using two oligonucleotidesdesigned to be able to hybridize to abutting sequences of a singlestrand of a target. One of the oligonucleotides is detectably labeled,and the other is biotinylated. If the precise complementary sequence isfound in a target molecule, the oligonucleotides will hybridize suchthat their termini abut, creating a ligation substrate. Ligation thenpermits the labeled oligonucleotide to be recovered using avidin oranother biotin ligand.

Ligase/Polymerase-mediated genetic bit analysis, described by Nifikorovet al. (supra), involves the immobilization of a first oligonucleotideto a solid substrate. The immobilized oligonucleotide is incubated witha sample containing the target molecule and with a secondoligonucleotide capable of hybridizing to the target molecule such thatthe two oligonucleotides are separated from one another by thepolymorphic site. A polymerase, a ligase and one deoxynucleosidetriphosphate are added. If the nucleotide at the polymorphic site iscomplementary to the deoxynucleoside triphosphate added, it will becomeincorporated by the polymerase and create a ligase substrate. Ligationcovalently couples the first oligonucleotide to the secondoligonucleotide indicating the identity of the polymorphic base.

In addition to methods that determine the actual sequence of apolymorphic site, other methods can distinguish between alternativesequences based on differing physical characteristics. These include,but are not limited to, dot-blot hybridization, sequencing byhybridization (SBH), denaturing HPLC, electrophoretic methods capable ofdistinguishing conformationally different nucleic acid molecules, orbinding to proteins capable of detecting mismatches between duplexedstrands of nucleic acids.

The dot blot method of genotyping with respect to a particularpolymorphism involves hybridization analysis using sequence-enriched oramplified DNA (i.e., reduced-complexity DNA) from an individual andoligonucleotide hybridization probes under conditions which allowdiscrimination of sequences based on single base pair differences. Thereduced-complexity DNA is fixed to hybridization membranes using methodsappropriate for the specific membrane type chosen (i.e., nitrocellulose,nylon, etc.). Kafatos et al., 1979, Nucl. Acids Res. 7:1541 describe amethod suitable for application of DNA samples to nitrocellulosemembrane involving alkaline denaturation and binding in high salt.Individual samples of the enriched, immobilized DNA are then hybridizedwith labeled oligonucleotides, each bearing one allelic form of thepolymorphic site, as described below. (A variation of this approach, the“reverse dot blot” method, uses labeled enriched DNA to probe specificpolymorphic oligonucleotides immobilized on a substrate.)

Filters bearing reduced-complexity DNA sequences are pre-hybridized in asolution consisting of 5×SSPE (1×SSPE is 180 mM NaCl, 10 mM NaH₂PO₄, 1mM EDTA), 5×Denhardt's solution (1×Denhardt's solution is 0.02% (w/v)polyvinylpyrrolidone, 0.02% (w/v) Ficoll, 0.02% (w/v) BSA, 0.2 mMTris-HCl, pH8.0, 0.2 mM EDTA), and 0.5% (w/v) SDS for at least 1 h at 55C. Radiolabeled probe, or probe detectably labeled by other means, isadded to the pre-hybridization mixture and incubated at 55° C. for 1 h.Each hybridized filter is then washed twice with 100 ml or more of2×SSPE, 0.1% SDS for 10 minutes at room temperature. High stringencywashes are then performed under temperature and salt conditions suchthat hybridization is only detected if the probe is 100% complementaryto the target sequence. That is, conditions are adjusted so that asingle base mismatch will abolish hybridization. Such conditions may bedetermined by one skilled in the art with a minimum of experimentationnecessary for any given polymorphism-containing oligonucleotide.Generally, the hybridization of shorter oligonucleotides (less than orequal to about 25 nt) is destabilized to a greater extent by single basechanges than the hybridization of longer ones. One method to achieve thenecessary level of specificity with a minimum of experimentation is tomaintain the temperature of the washes constant and vary the saltconditions. Lower salt concentrations are more stringent than highconcentrations. Specific examples of this type of hybridization beingused to determine the genotype of an individual with respect to apolymorphism, and the optimization of stringency are described byEhrlich et al., in the specification of U.S. Pat. No. 5,604,099.

Following washing, hybridized signal is detected by exposure to X-rayfilm, or by other appropriate means dependent on the type of label used(i.e., biotin, digoxigenin, etc.). Because hybridization only occurs ifthe probe and target sequences are 100% complementary, the presence of ahybridization signal with a particular probe directly indicates that theidentity of the polymorphic nucleotide is the complement of thecorresponding site on the individual probe used. This method may also beadapted to an array format for high throughput analyses.

SBH (Drmanac et al., 1993, Science. 260(5114): 1649; Drmanac et al.,1998, Nature Biotechnol., 16: 54) involves a strategy of overlappingblock reading. It is based on hybridization of DNA with the complete setof immobilized oligonucleotides of a certain length fixed in specificpositions on a support. The efficiency of SBH depends on the ability toeffectively sort out perfect duplexes from those that are imperfect(i.e. contain base pair mismatches). This is achieved by comparing thetemperature-dependent dissociation curves of the duplexes formed by DNAand each of the immobilized oligonucleotides with standard dissociationcurves for perfect oligonucleotide duplexes.

As another example of a method capable of detecting sequence differencesbased on differing physical characteristics, denaturing high performanceliquid chromatography can be used to screen samples for SNPs and othersequence variations (see Ophoff et al., 1996, Cell, 87: 543; Underhillet al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93: 196; Underhill et al.,1997, Virology, 237: 307; Liu et al., 1998, Nucleic Acids Res., 26:1396; and O'Donovan et al., 1998, Genomics, 52: 44). Alternatively,electrophoretic methods capable of detecting conformational differencesin nucleic acids may be used to distinguish polymorphic forms of nucleicacid molecules (see Keen et al., 1991, Trends Genet., 7: 5; White etal., 1992, Genomics, 12: 301). As another alternative, one may use aprotein capable of detecting mismatches between duplexed strands ofnucleic acid (see Parsons & Heflich, 1997, Mutat. Res., 374: 277).

Several embodiments of the invention utilize extension of an annealedprimer. This may be accomplished with any of a number oftemplate-dependent polymerases, including, but not limited to Klenow DNApolymerase, Taq DNA polymerase, and AMV or MML V Reverse Transcriptase.Conditions for primer extension using these polymerases are well knownand can be adjusted if necessary for a specific application by oneskilled in the art without undue experimentation. See, for example, thefollowing: 1) Klenow DNA polymerase—Kunkel et al., 1987, Meth. Enzymol.,154: 367; 2) Taq DNA polymerase Gelfand et al., 1990, PCR Protocols: AGuide to Methods and Applications, Academic Press, San Diego, Calif.;and 3) MMLV Reverse Transcriptase—Sambrook et al., 1989, MolecularCloning: A Laboratory manual, second edition, pp. 5.52-5.55, 8.11-8.17,Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

It should also be appreciated by one skilled in the art that the adaptermolecules that are operatively linked to the cleaved ends of nucleicacids may comprise a promoter sequence capable of initiating thesynthesis of RNA or DNA from the promoter site with an appropriatepolymerase. For example, the adapter may comprise a T7 RNA polymerasepromoter oriented so that transcription will proceed into the nucleicacid sample to which the adapter has been operatively linked (U.S. Pat.Nos. 5,716,785 and 5,891,636). When such adapter linked molecules areexposed to T7 RNA polymerase under the appropriate conditions, an RNAcopy of the nucleic acid sample will be created. The length of the RNAproducts can be controlled by adding chain-terminating ribonucleotidesubstrates into the reaction mixture in a concentration that willterminate an average transcript at a certain average length. The RNAproducts themselves, or a cDNA copy of the RNA products, can then beexamined for the presence of the polymorphism.

Conditions for in vitro transcription using, for example, T3, T7 or Sp6polymerase are well known and can be adjusted as necessary for aspecific application by one skilled in the art without undueexperimentation. See, for example, the following: 1) T3 RNApolymerase—Leary et al., 1991, Gene, 106:93; 2) T7 RNApolymerase—Bebendck & Kunkel, 1989, Nucleic Acids Res., 17:5408 andNoren et al., 1990, Nucleic Acids Res., 18: 83; and 3) Sp6 RNApolymerase—Melton et al., 1984, Nucleic Acids Res. 12: 7035.

EXAMPLES Example 1 Procedure for the Production of a Human/LambdaLibrary for Identification of Polymorphisms According to the Invention

The following contains an exact step by step description of theprocedure utilized in making the four libraries now being used for theidentification of SNPs. The procedure being utilized uses lambda ZAP IIas the cloning vector. Separate libraries have been made usingpBluescript as the cloning vector and other libraries have been madefrom lambda ZAP II using a slightly different procedure. From thestand-point of the procedure any cloning vector can be used if itcontains the suitable restriction enzyme sites. This procedure utilizesboth infrequent cutters of the human genome as well as frequent cuttersof the human genome.

The procedure is as follows:

1. Lambda Arm Production:

Left Arm Production

The left arm of lambda ZAP II is isolated by the following procedure.Thirty-five μg of lambda ZAP II DNA, in 35 μl of TE buffer (10 mMTris-HCl, pH 7.5, 1 mM EDTA-Na₂) is added to a tube containing 30 μl of10× Buffer 2 (250 mM NaCl, 100 mM Tris-HCl (pH 7.5), 100 mM MgCl₂, 100mM β-mercaptoethanol, 300 μg/ml Bovine Serum Albumin (BSA), finalconcentration). To the tube is added 205 μl of doubly distilled (ds)H₂0. The solution is mixed by inversion. Finally, 30 μl of therestriction enzyme Hind III (16 Units/μl, 480 U) are added. The solutionis then incubated at 37° C. Following 4 hours of incubation, thesolution is heated to 68° C. for 15 minutes (min). The solution is thenextracted twice with an equal volume of phenol followed by extractiononce with an equal volume of chloroform. After the extractions, the cutlambda DNA within the solution is precipitated by the addition of 2.5volumes of ethanol and incubation at minus 20° C. overnight or 20 min ina dry ice acetone bath. The precipitated cut lambda DNA is isolated bycentrifugation at 4° C. for 20 min at 14,000-×g. The pelleted cut lambdaDNA is then suspended in 75% ethanol and then re-pelleted. Finally, thepelleted cut lambda DNA is dissolved in 140 μl of dsH₂0.

The dissolved cut lambda DNA is then cut a second time with a differentrestriction enzyme by the following procedure. To the 140 μl of cutlambda DNA 10 μl of 10× universal buffer (1 M KOAc, 250 mM Tris-Acetate,pH 7.6, 100 mM MgOAc, 5 mM β-mercaptoethanol, 100 μg/ml BSA, finalconcentration) and 20 μg of the restriction enzyme Not I (8 U/μl, 160 U)is added. The solution is gently mixed by inversion and then incubatedat 37° C. for 1 h. Following the incubation, the solution is incubatedat 68° C. for 15 min. The solution is then extracted twice with an equalvolume of phenol followed by extraction once with an equal volume ofchloroform. After the extractions, the cut lambda DNA within thesolution is precipitated by the addition of 2.5 volumes of ethanol andincubation at minus 20° C. overnight or 20 min in a dry ice acetonebath. The pelleted cut lambda is then resuspended in low TE buffer (5 mMTris-HCl, pH 7.5 and 0.1 mM EDTA-Na₂, final concentration). Theresuspended cut lambda is then quantitated using a spectrophotometer,measuring absorbance at 260 nm and 280 nm. The left arm of the lambdavector is now ready for its role in the cloning of human genomic DNA.

Right Arm Production

The right arm of lambda ZAP II is isolated by the following procedure.Thirty-five μg of lambda ZAP II, in 35 μl of TE buffer is added to atube containing 60 μl of 10× universal buffer and 175 μl dsH₂0. Aftermixing the solution by inversion, 30 μl of the restriction enzyme Mlu I(32 U/μl, 960 U) are added. The solution is then incubated at 37° C. for4 h. After the incubation, the solution is heated to 68° C. for 15 min.

The solution is then extracted twice with an equal volume of phenolfollowed by extraction once with an equal volume of chloroform. Afterthe extractions, the cut lambda DNA within the solution is precipitatedby the addition of 2.5 volumes of ethanol and incubation at minus 20° C.overnight or 20 min in a dry ice acetone bath. The precipitated cutlambda DNA is isolated by centrifugation at 4° C. for 20 min at14,000-×g. The pelleted cut lambda DNA is then suspended in 75% ethanoland then re-pelleted. Finally, the pelleted cut lambda DNA is dissolvedin 170 μl of dsH₂O.

The dissolved cut lambda DNA is then cut a second time with a differentrestriction enzyme by the following procedure. To the 170 μl of cutlambda DNA 20 μl of 10× universal buffer and 10 μl of the restrictionenzyme EcoRI (24 U/μl, 240 U) is added. The solution is gently mixed byinversion and then incubated at 37° C. for 1 h. Following theincubation, the solution is incubated at 68° C. for 15 min.

The solution is then extracted twice with an equal volume of phenolfollowed by extraction once with an equal volume of chloroform. Afterthe extractions, the cut lambda DNA within the solution is precipitatedby the addition of 2.5 volumes of ethanol and incubation at minus 20° C.overnight or 20 min in a dry ice acetone bath. The pelleted cut lambdais then resuspended in low TE buffer. The resuspended cut lambda is thenquantitated using a spectrophotometer, measuring at 260 nm and 280 nm.The right arm is now ready for its role in the cloning of human genomicDNA.

Preparation of human Genomic DNA for Cloning

Human genomic DNA is prepared for cloning by the following procedure.Twenty μg of human genomic DNA in 20 μl of TE are added to a tubecontaining 10 μl of universal buffer and 60 μl of dsH₂O. Ten μl of therestriction enzyme EcoRI (24 U/μl, 240 U) are added to the tube. Thetube is then incubated at 37° C. for 2 h. After the incubation, thefollowing is added to the tube: 42 μl of dsH₂O, 5 μl of 10× universalbuffer and 3 μl (171 U) of calf intestinal alkaline phosphatase. Thetube is then incubated for an additional 30 min at 37° C. Following theincubation, the solution is heated to 68° C. for 15 min.

The solution is then extracted twice with an equal volume of phenolfollowed by extraction once with an equal volume of chloroform. Afterthe extractions the cut human genomic DNA within the solution isprecipitated by the addition of 2.5 volumes of ethanol and incubation atminus 20° C. overnight or 20 min in a dry ice acetone bath. Theprecipitated, cut human genomic DNA is isolated by centrifugation at 4°C. for 20 min at 14,000×g. The pelleted, cut human genomic DNA is thensuspended in 75% ethanol and then re-pelleted. Finally, the pelleted,cut human genomic DNA is dissolved in 70 μl of dsH₂O.

The dissolved cut human genomic DNA is then cut a second time with adifferent restriction enzyme by the following procedure. To the 70 μl ofcut lambda DNA 20 μl of 10× universal buffer and 10 μl of therestriction enzyme Not I (8 U/μl, 80 U) are added. The solution isgently mixed by inversion and then incubated at 37° C. for 2 h.Following the incubation, the solution is incubated at 68° C. for 15min.

The solution is then extracted twice with an equal volume of phenolfollowed by extraction once with an equal volume of chloroform. Afterthe extractions, the cut human genomic DNA within the solution isprecipitated by the addition of 2.5 volumes of ethanol and incubation atminus 20° C. overnight or 20 min in a dry ice acetone bath. Thepelleted, cut human genomic DNA is then resuspended in low TE buffer.The resuspended, cut human genomic DNA is then quantitated using aspectrophotometer, measuring absorbance at 260 nm and 280 nm. Thedigested Human genomic DNA is now ready for its role in the cloning intothe vector.

Ligation of the Human Genomic DNA into the Two Lambda ZAP II Arms.

A total of 1 μg of lambda vector arms at a ratio of 1 to 15, left arm toright arm, is mixed with 0.1 μg of the double cut human genomic DNA, 0.5μl of 10× ligase buffer (500 mM Tris-HCl, pH 7.5, 70 mM MgCl₂, 10 mMdithiothreitol), 1 mM rATP, and 0.5 μl of T4 ligase (2 U; Stratagene) ina total volume of 5 μl. The solution is then incubated at 4° C.overnight.

Packaging of the Human/Lambda ZAP II Library.

All procedures used for the packaging of the lambda/human DNA isdescribed within the directions and package insert for the kit asprovided by Stratagene.

Preparation of Host Bacteria

The bacterial glycerol stock contained within the kit is used to streakLuria-Bertani agar (LB, Bacto-tryptone 10 g/l, Bacto-yeast extract 5g/l, NaCl 5 g/l and Bacto-agar 15 g/l). The bacterial glycerol stockcontains the Eschericia coli strain VCS257. The streaked plates areincubated overnight at 37° C. A single colony is then used to inoculateLB media supplemented with 10 mM MgSO₄ and 0.2% (w/v) maltose. Theinoculated medium is incubated at 37° C. with shaking for 4-6 h, notpast an OD₆₀₀ of 1.0. After incubation, the cells are pelleted bycentrifugation at 500-×g for 10 min. The cells are then gentlyresuspended in half their original volume in 10 mM MgSO₄. The cells arenow ready for use in the packaging protocol.

Packaging Protocol

The packaging extracts provided with the kit are removed from the minus80° C. freezer and placed on dry ice. The tube is then quickly thawed byholding the tube between one's fingers. The human/lambda ZAP II DNAligation mixture (1.7 μl) is added to the packaging extract. Thepackaging mixture is then stirred with a pipette tip to insure evenmixing. The tube is then quickly spun (3-5 seconds). The tube is thenincubated at room temperature (22° C.) for 2 hours. After the incubation500 μl of SM buffer (100 mM NaCl, 8.11 mM MgSO₄, 50 mM 1M Tris-HCl (pH7.5), 0.01% gelatin) is added to the tube. Twenty μl of chloroform isthen added to the tube and the contents are mixed. The tube is spunbriefly and the supernatant is transferred to a fresh tube. The phagecontained within the supernatant are now ready for further usage.

These procedures have been carried out for four separated human genomes.The resulting four human/lambda libraries are being utilized for thedetection of SNPs.

Example 2 Procedure by which the Human/Lambda Phage Library is Utilizedto Detect Polymorphisms

Following is a step by step description of the procedure for thediscovery of SNPs within the human genome according to the invention.

Plating and Picking of the Human/Lambda Library

Plaque Plating

XL 2-Blue MRF Eschericia coli cells are maintained as a stock bystreaking the cells on LB agar for single colonies. Single colonies areused to inoculate 3 ml LB media. The bacteria are grown at 37° C. for 6hours with gentle shaking. After incubation, the bacteria are pelleted(500-×g) and then resuspended in 1.5 ml of 10 mM MgSO₄. These cells arethen used for transduction by the human/lambda library. Utilizing theappropriate library phage and the bacterial cells suspended in 10 mMMgSO₄, plaque plates are generated as follows: 0.1 ml of the bacterialsuspension is mixed with enough library phage to yield between 100 and300 plaques per 150 mm agar plate. The mixture is incubated at roomtemperature for 5 min. After the incubation 2.5 ml of pre-warmed LB (37°C.) is added followed by 2.5 ml of molten (45° C.) top agar (10 g/lBacto-tryptone, 5 g/l Bacto-yeast extract, 5 g/l NaCl and 7 g/lBacto-agar). This mixture is then immediately poured over a pre-dried LBagar plate (predried for 6-8 h at 37° C.). Initially the plate isincubated right side up at room temperature. After 30 min the plate theplate is placed in a 37° C. incubator bottom side down and incubated fora minimum of 10 h. Plaques begin to appear at 4 hrs. Plates areincubated for no longer than 8 hrs before they are removed from theincubator and placed at 4° C.

Picking Plaques from the Plated Library

Plaques are picked by coring the middle of the plaque using a P200pipetter and widebore P200 ART tips. Only the top agar is cored and usedto make the plaque/phage stocks. Once the plaque is cored, the agar plugis placed in 70 μl of SM buffer containing 5 μl of chloroform.Plaque/phage stocks are maintained in 96-well polystyrene plates.Individual wells are capped and the plate is wrapped in Parafilm™ andstored at 4° C. until they are used. Once a plate has been processed foradditional procedures Dimethyl Sulfoxide (DMSO) is added to every wellto final concentration of 10%. The plates are then stored at −80° C.

Plague/phage Polymerase Chain Reaction

The inserted cloned human DNA is next amplified by PCR. Reactions areset up in 96-well formats that replicate the 96-well format of theplaque/phage stock plate. The PCR reactions are 25 μl in total volumeand consist of the following 2 μl of plaque/phage stock, 2.5 U of PfuTurbo, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dTTP, 0.1 mM dGTP, 0.1 mM7-deaza dGTP, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM Ammoniumsulfate, 0.1% Triton X-100, 0.1 mg/ml BSA and 25 μM primers. The primersbeing utilized now for PCR are vector specific primers that allowamplification of both the inserted human DNA as well as fragments of thevector. Plaque/phage stocks are run in duplicate with the onlydifference being the addition of 5% DMSO to one of the duplicate wells.The PCR cycling conditions are as follows: 1 cycle of 98° C. for 3 min:followed by 2 cycles 98° C. for 2 min, 55° C. for 50 sec, 75° C. for 7min followed by 29 cycles of 95° C. for 50 sec, 58° C. for 50 sec, 75°(C for 7 min, followed by a final elongation at 75° C. for 15 min.

Following PCR, the plus and minus DMSO plates are consolidated into oneplate and stored at 4° C.

PCR Reaction Cleanup

PCR products are purified using a 96-well format by employing binding toglass fiber filters in a high salt solution. To each well an equalvolume (approximately 50 μl) of binding buffer (4 M guanidineisothiocyanate in 100 mM Tris-HCl (pH 6.4)) is added. The solution(binding buffer and PCR product) is then transferred to the appropriatewell of the PCR purification plate. A vacuum is then applied to the PCRpurification plate (400 mbar) until all of the liquid has been removedfrom the wells. The vacuum is then applied for an additional 5 min.After the 5 min. each well is washed with 750 μl of wash buffer (75%ethanol, 2 mM Tris-HCl (pH 6.5), 10 mM NaCl). Vacuum is then againapplied to the plate until the wells are dry. Vacuum is applied for anadditional 15 min. The PCR purification plate is then centrifuged at1000-×g for 10 min. The purified PCR product is eluted from the filterby the addition 50 μl of 10 mM Tris-HCl (pH 8.5) to the filter,incubating at room temperature for 5 min, placing a 96-well collectionplate underneath the PCR purification plate and centrifuging the platesat 1000×g for 10 min.

Quantitation of the Purified PCR Product

In order to determine which PCR reactions have produced product and toquantitate the amount of purified PCR product present, the fluorescentdye picogreen is used. The procedure is as follows: in a 96-well plate 5μl of each purified PCR product is placed in the appropriatecorresponding well which contains 95 μl of TE. To each appropriate well100 μl of picogreen, which has been diluted to 200 with TE, is added.The plate is then incubated in the dark for 10 min and then read withthe proper spectrofluorometer at the appropriate wavelengths forexcitation and emission. Utilizing the proper standards, wells thatcontain a PCR product can be consolidated into another 96-well plate.

Sequencing of PCR Products

Consolidated, purified PCR products are then cycle-sequenced usingBigDye Terminator chemistry (Perkin Elmer/Applied Biosystems). Othertypes of sequencing chemistries are also compatible with this process.Sequencing is done in a 384 well format and is carried out as follows:one or 2 μl of the purified PCR product are mixed with 4 μl of theBigDye Ready Reaction Mix, 1 μl of the sequencing primer (either T7 orT3) and enough dsH₂O to bring the total reaction volume to 10 μl. Cyclesequencing is then initiated using the following parameters: 25 cyclesof 96° C. for 25 sec, 45° C. for 45 sec and 60° C. for 4 min 25 sec.Samples are then precipitated by the addition of 2.5 volumes of 100%ethanol. The samples are incubated at room temperature for 15 min andthen centrifuged at 3000×g for 30 min. The plate is then inverted andplaced on top of a paper towel and re-centrifuged at 400×g for 1 min.The plates are then allowed to air dry for 15 min at room temperature.The pelleted cycle-sequencing product is then dissolved in 2.5 μl 80%formamide containing a tracking dye and 5 mM EDTA-Na₂. Samples aredenatured at 96° C. for 2 min and then placed on ice before they areloaded onto the sequencing gel. Sequencing gels are set up and runaccording to standard procedures specified by the manufacturer PerkinElmer/Applied Biosystems.

It should be noted that the process of SNP discovery is not limited tothe procedures described above.

Example 3 Enriching for and Identifying a Nucleic Acid SequenceDifference with Respect to a Reference Sequence

In order to enrich a nucleic acid sample for a subset of nucleic acidmolecules bearing a marker sequence, that sample is reacted with asequence-specific binding activity under conditions that permit specificbinding. The sequence specific binding activity can be any activity thatbinds to a particular sequence or sequence motif. Examples include, butare not limited to transcription factors or their DNA binding domains(e.g., Fos and Jun, see Cohen & Curran, 1990, Oncogene, 5: 929) proteinswith zinc-finger DNA binding domains (Cohen et al., 1992, Science, 257:1951), restriction endonuclease DNA recognition domains,sequence-specific antibodies (Erez-Alon et al., 1998, Cancer Res., 58:5447), oligonucleotides complementary to an adapter ligated to apopulation of DNA molecules, nucleic acid molecules, aptamers (Hale &Schimmel, 1996, Proc. Natl. Acad. Sci. U.S.A., 93: 2755; Feigon et al,1996, Chem. Biol., 3: 611), peptide nucleic acid molecules (Kuhn et al.,1999, J. Mol. Biol., 286: 1337; Ratilainen et al., 1998, Biochemistry,37: 12331), peptides (Banks et al., 1999, J. Biol. Chem., 274: 16536)and affinity resins that recognize DNA having a particular G+C contentor methylation status.

The binding conditions for the sequence specific binding activity usedaccording to the invention are known by those skilled in the art. Thatis, the binding conditions will vary with the identity of the particularsequence-specific binding activity selected for use in the method of theinvention, but in order for a particular sequence-specific bindingactivity to be selected for use in this method the conditions for itssequence-specific nucleic acid binding are known in the art.

For example, a DNA binding activity such as the NF-κB p50/p65 complexmay be used to select molecules bearing NF-κB recognition sequencesunder binding conditions as used for protein:nucleic acid binding in thewell-known electrophoretic mobility shift assay (Kunsch et al. 1992,Mol. Cell. Biol., 12: 4412).

As another example, a nucleic acid may be used as a sequence-specificbinding activity, according to the invention, under conditions known tothose skilled in the art to permit specific hybridization or annealingto its complementary sequence. The nucleic acid used as a sequencespecific binding activity according to the invention may be adouble-stranded molecule having an overhang allowing annealing tomolecules with a complementary overhang, or it may be a single-strandedoligonucleotide that hybridizes at a specific marker sequence on asub-population of nucleic acids in the sample.

In cases where a double-stranded nucleic acid sequence with a specificoverhang is used as a sequence-specific binding activity according tothe invention, the interaction may be stabilized by the activity of aligase, such as T4 DNA ligase, under conditions known in the art.

Conditions for hybridization or annealing of a single-stranded nucleicacid as a sequence-specific binding activity are similar to those usedfor annealing of primers in PCR applications. For example, standard PCRconditions call for annealing primers at a temperature 2° C. to 5° C.below the calculated T m for a given primer in a buffer comprising 50 mMKCl, 10 mM Tris-HCl (pH 8.4) and 100 mg/ml gelatin (see Gelfand et al.,supra). The annealing temperature for a given nucleic acid with itscomplement may be estimated according to the following formulae, whichaccount for the length, G+C content, and salt conditions in thereaction. For oligonucleotides shorter than 14 bases, use the formulaT_(m)=2° C. (A+T)+4° C. (G+C); for oligonucleotides 14 bases and longer(up to 60-70 nucleotides), use the formulaT_(m)=81.5+16.6(log₁₀[Na⁺])+0.41 (% G+C)−(600/n), where “n” is the chainlength. For probes longer than 70 bases, use the formulaT_(m)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(675/n). Due to the effects ofbase stacking, near neighbor effect and buffering capacity, which willvary with the exact oligonucleotide sequence, these formulae give only aclose approximation of T_(m). However, it is well within the capacity ofone of ordinary skill in the art to tailor temperatures to a particularoligonucleotide sequence, without undue experimentation, using theseformulae as a starting point. Alternatively, a more precise T_(m)determination may be made using the method of Arnold et al., (U.S. Pat.No. 5,283,174).

The binding activity may be free in solution or attached to a solidsupport, such as beads or a nylon or nitrocellulose membrane tofacilitate the physical separation of protein:nucleic acid complexesfrom unbound nucleic acids. One of skill in the art may attach bindingactivities such as proteins (including antibodies), peptides, nucleicacids, aptamers or peptide nucleic acids to solid supports for use withthe method of the invention. Attachment may be direct, as is possiblefor some activities on different types of solid supports (e.g., proteinor nucleic acid binding to nitrocellulose or nylon membranes), orindirect, mediated for example by an antibody specific for the sequencespecific binding activity or by a molecule, such as streptavidin, whichrecognizes a labeling moiety, such as biotin, on the sequence-specificbinding activity.

As a specific example of the use of a sequence-specific binding activityto enrich for and identify a nucleic acid sequence difference withrespect to a reference sequence, one may use the NF-κB p50 DNA bindingprotein to enrich for DNA molecules containing the consensus sequence5′-GGPuNNPyPyCC-3′, and the enriched population may be analyzed forsequence differences. To do this, one must take the following steps:

1. Enrich a Genomic DNA Sample for Molecules Bearing the NF-κB ConsensusSequence.

DNA at least 20 μg is incubated with beads bearing immobilized NF-κB p50protein according to the binding conditions of Kunsch et al. (Kunsch etal., supra). The unbound DNA is removed by washing the beads three timeswith binding buffer, at ten times the packed volume of the beads perwash. Alternatively, the DNA-binding protein-bearing beads may be madeinto a column, with DNA being passed over the column in binding buffer.Unbound sequences are then removed by passing several void volumes ofbinding buffer without DNA over the column.

2. Detect a Nucleic Acid Sequence Difference with Respect to a ReferenceSequence.

The bound nucleic acid is eluted, using for example, 300 mM sodiumacetate, pH 5.0. Under these conditions, the eluted DNA may be readilyconcentrated by ethanol precipitation. The DNA, thus enriched formolecules bearing the NF-κB consensus sequence is then ready for furtherenrichment or for analysis with respect to a reference sequence by anyof the genotyping methods described elsewhere herein (e.g., DNAsequencing (including primer-guided microsequencing and minisequencing),Exonuclease-resistance, extension in solution using ddNTPs, GBA™,ligase-polymerase GBA, OLA, dot-blot/allele-specific hybridization, SBH,denaturing HPLC, electrophoretic methods capable of distinguishingconformational differences between nucleic acids, or binding of proteinscapable of detecting mismatches between duplexed strands of nucleicacids. The enrichment protocol will increase the sensitivity and reducethe background in each of these methods.

Example 4 Enriching for and Identifying a Nucleic Acid SequenceDifference with Respect to a Reference Sequence

In this example, the sequence specific cleavage agents NotI and EcoRIare used to select molecules bearing sequences near the infrequentlyoccurring NotI sites in the genome. To do so, one will take thefollowing steps:

1) Cleave a DNA sample with NotI and EcoRI. The DNA may be genomic or,for example, cDNA made by reverse-transcription of a total RNA or mRNAsample. Cleavage is performed according to the conditions specified bythe supplier of the enzymes.

2) Ligate the sub-population of molecules having both a NotI- and anEcoRI-cleaved end to molecules facilitating their replication. Themolecules facilitating replication of the linked DNA may be either anappropriate plasmid cleaved with both Not and EcoRI (for example,pBluescript II SK, Stratagene), or they may be double-strandedoligonucleotides with overhangs allowing annealing and ligation to theNotI and EcoR ends of the cleaved DNA. Such oligonucleotides may be freein solution or they may be immobilized on a solid support.

Alternate replication vectors may also be used, such as bacteriophagelambda DNA. For example, Lambda ZAP (Stratagene) is digested with Not Iand EcoR I and ligated with the DNA sample. There are several techniquesthat can be used to enrich for vector containing only fragments with NotI and EcoR I ends. For example, the sample DNA may be cleaved with EcoRI first, then treated with an alkaline phosphatase, such as calfintestinal alkaline phosphatase (Boehringer Mannheim) according to themanufacturer's instructions, to remove 5′ phosphates from the sampleDNA, then digested with Not L. The Not I/EcoR I digested fragments willbe able to ligate with the vector DNA, but EcoR I/EcoR I sample DNAfragments will not be able to ligate with each other, thus reducing thefrequency of inserts containing EcoR I/EcoR I fragments.

Another method is to use excess vector having an EcoR I compatible end.For example, Lambda ZAP DNA may be cleaved with Not I and Hind III. Suchdouble digestion leaves a replication-competent left arm of lambda, butcleaves the right arm into multiple fragments. Another preparation ofLambda ZAP DNA may be cleaved with EcoR I and Mlu I. Such doubledigestion leaves a replication-competent right arm of lambda, butcleaves the left arm into multiple fragments. Since Mlu I cleaves lambdamany times in the left arm, and Hind III cleaves lambda many times inthe right arm, such double-digested preparations do not efficientlyproduce replication-competent lambda genomes upon exposure to T4 DNAligase. Moreover, if both double-digested preparations are mixedtogether and exposed to T4 DNA ligase, replication-competent lambdagenomes are still rare. However if such double-digested preparations aremixed together along with sample DNA that has been digested with Not Iand EcoR I, then DNA fragments that have been cleaved at one end by NotI and at the other end by EcoR I will be able to ligate with thefunctional Not I-cleaved left arm from one lambda preparation and ligatewith the functional EcoR I-cleaved right arm from the other lambdapreparation to produce replication-competent lambda genomes withreasonably high efficiency. This is provided that the Not I/EcoR Icleaved sample DNA fragments are of appropriate size for lambdareplication, which for Lambda ZAP would be fragments up to about 9kilobases in length. If a fragment with Not I cleavages at both ends, ora fragment with EcoR I cleavages at both ends ligates with one of thelambda arms, it would not be able to ligate with the opposite lambdaarm, and thus would form a replication-incompetent product. Since EcoR Icleaves human DNA about 25 times more frequently than does Not I, onecan add about 25 times more lambda right arms, as compared with leftarms (molar ratio), to the ligation reaction. This will increase theprobability that each Not I/EcoR 1-cleaved sample fragment will ligatewith an EcoR I-cleaved right arm rather than another EcoR I-cleavedsample DNA fragment. Such unwanted ligation of EcoR I-cleaved samplefragments can also be reduced by treatment with an alkaline phosphatase,such as calf intestinal alkaline phosphatase.

Another method to enrich for ligated molecules containing only NotI/EcoR I doubly cleaved sample fragments, is to cleave the sample DNAfirst with Not I, then ligate the cleaved DNA with a plasmid vector thatcomprises a Not I and an EcoR I site, but has been cleaved only with NotI. After ligation, the DNA is cleaved with EcoR I, which cleaves boththe vector DNA and the sample DNA. Following inactivation of the EcoR Iactivity, the sample is diluted and ligated using T4 DNA ligase. DilutedDNA preferentially circularizes as opposed to forming bimolecularreactions. Thus there will be a large fraction of circularized plasmidscontaining NotI/EcoR I cleaved sample DNA.

3) Replicate the Linked Molecules Generated in Step 2.

When the molecules facilitating replication of the linked DNA comprise aplasmid, this comprises the steps of transforming competent host cells(Stratagene) and selecting for transformants according to standardmethods.

When the molecules facilitating replication of the linked DNA comprise alambda genome, this comprises the steps of transfecting host cells andselecting for growth of lambda bacteriophage according to standardmethods. Lambda bacteriophage packaging extract (Gigapack™, Stratagene)can be used to greatly increase the efficiency of lambda DNAtransfection.

When the molecules facilitating replication of the linked DNA areoligonucleotides or adapters, this comprises the annealing of a primercomplementary to one strand of the oligonucleotides or adapters ligatedto the NotI cleaved or NotI/EcoRI cleaved DNA fragments and polymerizingthe complementary strand of the ligated molecule with atemplate-dependent DNA polymerizing enzyme (e.g., Klenow DNA polymeraseor Taq DNA polymerase; conditions for primer extension with theseenzymes and others are well known in the art). Examples of suchreplication can be found in Lisitsyn et al., 1993, Science, 259: 946;Hubank & Schatz, 1994, Nucl. Acids Res., 22: 5640; Hou et al., 1996,Nucl. Acids Res., 24: 2196; Suzuki, et al., 1996, Nucl. Acids Res., 24:797; and Lukyanov et al., 1996, Nucl. Acids Res., 24: 2194. In order toenrich for those sequences near NotI sites, one uses a primercomplementary to the adapter ligated to the Not ends of the cleavedpopulation. The degree of enrichment may be enhanced by repeating thepolymerization reaction. In this regard, thermostable polymerases suchas Taq DNA polymerase have the advantage of permitting cycles ofannealing and extension, which increases the degree of enrichment witheach cycle. Alternatively, strand displacing polymerases can be used toproduce multiple copies of the linked DNAs at a single temperature (U.S.Pat. No. 5,744,311).

Alternatively, if a double-stranded oligonucleotide ligated to one endof the cleaved molecules generated in step (1) comprises atranscriptional promoter, such as the bacteriophage T7 promoter, thereplication step may comprise the steps of adding RNA polymerase (e.g.,T7 polymerase) and ribonucleotides under conditions allowing RNApolymerization from the ligated promoter. Conditions for such in vitrotranscription are well known in the art (U.S. Pat. Nos. 5,891,636 and5,716,785), and the transcripts may be labeled if necessary (labelsinclude, but are not limited to a fluorescent molecule, radioactivemolecule, hapten, or biotin). Under optimal conditions, up to 700 molesof transcript can be generated per mole of DNA template, thereby givingas much as a 700-fold enrichment for sequences bearing a ligatedpromoter.

4) Detect One or More Nucleic Acid Sequence Differences with Respect toa Reference Sequence in the Replicated Population of Molecules.

Detection of nucleic acid sequence differences in the enrichedsub-population generated in step (3) is then achieved using any of thegenotyping methods described herein. These methods include, for example,DNA sequencing (including primer-guided microsequencing andminisequencing), exonuclease resistance, extension in solution usingddNTPs, GBA™, ligase-polymerase GBA, OLA, dot-blot/allele-specifichybridization, SBH, denaturing HPLC, electrophoretic methods capable ofdistinguishing conformational differences between nucleic acids, orbinding of proteins capable of detecting mismatches between duplexedstrands of nucleic acids. The enrichment protocol will increase thesensitivity and reduce the background in each of these methods.

When the in vitro replication system involves transcription from aligated bacterial promoter, allele-specific (dot-blot) hybridization maybe used to detect sequence differences. Alternatively, the ribonucleaseprotection assay may be used to detect sequence differences in RNAmolecules. The method of RNAse protection is well known in the art, andseveral companies sell kits for the method, including Ambion (RPAII™kit, Cat. # AM-1410) and Pharmingen (RiboQuant™ kit). The methodinvolves synthesis of an RNA probe from a plasmid bearing abacteriophage promoter and an insert containing the reference sequence.The RNA probe is then hybridized with the RNA generated in theenrichment protocol. A ribonuclease capable of cleaving single strandedor mismatched duplexes, but not perfectly matched duplexes, is thenadded. Cleaved duplexes provide evidence of mutations in the sample RNAas compared with the reference RNA. Typically these cleavage productsare identified by gel electrophoresis.

Alternatively, the RNA can be sequenced directly using Sanger sequencingand the enzyme reverse-transcriptase. The RNA may also be converted tocDNA, and then the cDNA may be sequenced.

Example 5 Enriching for and Identifying a Nucleic Acid SequenceDifference with Respect to a Reference Sequence

In this example, the sequence specific cleavage agents NotI and EcoRIare used to select molecules bearing sequences near the infrequentlyoccurring NotI sites in the genome. To do so, one will take thefollowing steps:

1) Cleave a DNA sample with NotI and EcoRI.

The DNA may be genomic or, for example, cDNA made byreverse-transcription of a total RNA or mRNA sample. Cleavage isperformed according to the conditions specified by the supplier of theenzymes or as known in the art.

2) Link the Sub-Population of Molecules Having a NotI End to MoleculesFacilitating their Separation.

A useful reference for this procedure is Hultman & Uhlen, 1994. JBiotechnol, 30:35: 229. The molecules facilitating separation of themolecules with NotI ends may be double-stranded oligonucleotides withNotI-compatible overhangs allowing annealing of the cleaved DNA. Theannealed NotI fragments are then ligated to the double-strandedoligonucleotides with ligase under standard conditions. This processwill link those sequences near NotI sites to molecules facilitatingtheir separation from those sequences further than the nearest EcoRIrecognition sequence from a NotI recognition sequence.

3. Separate the Linked Molecules.

The oligonucleotides may be bound to a solid support at any pointbefore, during or after annealing and ligation of the DNA fragments. Inany case, the fragments linked to the oligonucleotides are separatedfrom those not linked to the oligonucleotides by washing the solidsupport after linkage of the oligonucleotides to the population offragments. Wash buffer may be a standard buffer such as TE (10 mM TrispH 8.0, 1 mM EDTA), or any buffer compatible with the solid support andmethod of oligonucleotide linkage to it.

4. Detect One or More Sequence Differences in the Bound Population withRespect to a Reference Sequence.

Methods of detecting sequence differences appropriate for this enrichedpopulation include DNA sequencing, denaturing HPLC, electrophoresiscapable of differentiating conformational differences in nucleic acids,and binding to a protein capable of detecting mismatches betweenduplexed strands of nucleic acid.

Example 6 Enriching for and Identifying Nucleic Acid SequenceDifferences with Respect to a Reference Sequence

In this example, a sample of nucleic acid is treated to generatefragments that are then bound to a sequence-specific binding activity toeffect an enrichment for either those molecules bearing or lacking thesequence bound by that activity. To do so, one must perform thefollowing steps.

1) Fragment a Nucleic Acid Sample to the Chosen Approximate AverageFragment Length.

A nucleic acid sample may be fragmented to facilitate the enrichment formolecules bearing a particular marker sequence. Fragmenting may beaccomplished by physical means, such as shearing, or by cleavage with anagent such as a restriction endonuclease. While other cleavage agentsare useful according to the invention, restriction endonucleases areparticularly useful for several reasons. First, the frequency of cuttingfor a particular restriction endonuclease, and thereby the averagefragment length generated by digestion of a genomic or other DNA sample,is often known or predictable based on the length of the recognitionsequence and the nucleotide makeup of the recognition sequence (G/C orA/T rich, for example). Similarly, the average fragment length for acombination of two or more restriction endonucleases may be predicted.Therefore, DNA may be fragmented to a selected average fragment lengthby selection of two or more restriction endonucleases of the appropriateknown cutting frequencies. It should be noted that there are cases inwhich a restriction endonuclease will not generate fragments of a sizepredicted on the basis of the base composition of its recognitionsequence. For example, if a recognition sequence for a particular enzymeoccurs in a highly repeated segment of DNA, the average number and sizeof the fragments will be altered relative to a similar sequence notoccurring in a repeated element. In practice, the average size offragments generated by a given restriction endonuclease may be estimatedby examination of fragments after electrophoretic separation on a gel.For additional information on the distribution and size fractionation ofrestricted genomic DNA fragments, see Gondo, 1995, Electrophoresis, 16:168.

Another advantage of fragmenting with restriction endonucleases is thatmany of the known enzymes cleave so as to generate an overhang on onestrand. That overhang may be exploited in subsequent steps. For example,the portion of the cleaved population bound to a sequence specificbinding activity may be ligated or annealed to a nucleic acidmolecule-that permits its cloning to form a library of sequences.Alternatively, the cleaved, bound population may be ligated or annealedto a primer that permits its replication or transcription. Thereplication or transcription of the molecules bound to thesequence-specific binding activity will further enrich the populationand facilitate the detection of sequence differences in the bound subsetof nucleic acid molecules with respect to a reference sequence.

2) Physically Separate a Subset of the Nucleic Acid Fragments Generatedin Step (1) Based on the Presence or Absence of a Particular NucleicAcid Sequence.

Fragments bearing a given sequence or sequence motif may be separatedfrom those lacking such a sequence with a sequence-specific bindingactivity under conditions compatible with sequence-specific binding bythat activity (see, for example, Example 3). It should be understoodthat either the population bearing or the population lacking theparticular sequence or sequence motif bound by a sequence-specificbinding activity, or both, may be further analyzed as enrichedpopulations.

3) Link the Subset of Nucleic Acid Molecules Physically Separated inStep (2) to Molecules Facilitating the Replication of the Subset.

When the cleavage method used is random, such as, for example, physicalshearing, a method such as that taught by Andersson et al. can be usedto link the subset of molecules to molecules facilitating theirreplication (Andersson et al., 1996, Anal. Biochem. 236: 107) Briefly,the method involves enzymatic repair (blunting) of the ends of thesheared molecules, followed by ligation to adapters with 12 bpoverhangs. The oligonucleotide adapters used are non-phosphorylated,thus preventing formation of adapter dimers and ensuring efficientligation of fragments to the adapters. The ligated fragments are thenannealed to a modified M13 vector with ends complementary to the adapteroverhangs and transformed into bacteria without ligation.

4) Replicate the Subset of Molecules Linked in Step (3).

Linked molecules may be replicated as in Example 4, section 3.

5) Detect a Sequence Difference with Respect to a Reference Sequence.

Detection of sequence differences with respect to a reference sequencemay be performed using the same methods indicated in Example 4, section4, above, or any suitable method known in the art.

Example 7 Enriching for and Identifying Nucleic Acid SequenceDifferences with Respect to a Reference Sequence

In this example, the sequence specific binding activity is one or moreoligonucleotide primers that hybridize to a sequence that occurs atleast twice, but can occur for example 3, 4, 5, 10, 20, 50, 100, 1000,10,000, 25,000, 50,000 or even 100,000 times or more per genome (seeAP-PCR, Welsh & McClelland, 1990, supra; and RAPD, Williams et al. 1990,supra). To enrich for and identify nucleic acid sequence differenceswith respect to a reference sequence according to this method, one mustperform the following steps.

1) Hybridize a Nucleic Acid Sample from One or More Individuals withOligonucleotide Primers.

Conditions for annealing primers, particularly as used in PCRapplications, are well known in the art (Gelfand et al., supra). Aprimer for this particular method may be as short as about five to eightnucleotides, although longer primers are permissible or even preferredin some situations (see below). The number of extension products is afunction of the efficiency of annealing under a given set of conditions,and can be manipulated by one of skill in the art to give a desiredapproximate number of extension products. For example, in general, theannealing temperature is inversely proportional to the number ofextension products for a given primer on nucleic acid from a givenspecies. Therefore, the higher the annealing temperature, the fewer theproductive extension events. Other factors, such as the makeup of thepolymerization buffer or the presence of chain-terminating nucleosideanalogs can also be varied to change the makeup of the extendedpopulation (see below).

2) Extend the Annealed Oligonucleotide Primers to Form an EnrichedCollection of Replicated Molecules.

Extension may be performed with a template-dependent DNA polymerase suchas Taq DNA polymerase or Klenow DNA polymerase. Alternatively, extensionof an oligonucleotide annealed to an RNA template may be extended withreverse transcriptase.

Annealing and extension may be repeated to increase the degree ofenrichment with any of the enzymatic systems described. As noted,however, Taq DNA polymerase has the advantage of allowing multiplecycles of annealing and extension without requiring repeated enzymeaddition. It is also noted that the processivity of Taq DNA polymeraseis sensitive to the concentration of Mg⁺² in the reaction, and can bevaried by one skilled in the art to vary the characteristics of theextended products.

Under some circumstances (e.g., when one wishes to further limit thecomplexity of the resulting population, or when one wishes to generatean incomplete extension product), one may add a chain-terminatingnucleoside analog to the extension mixture at a concentration thatlimits the length of the average extension product. Within thisembodiment of the invention, one may wish to limit the length of theaverage extension product to any length between about 500 and 5000 nt.One of skill in the art may determine the concentration ofchain-terminating nucleoside analog to add to achieve a given desiredaverage extension product length with a minimum of experimentation.

The extension products may be detectably labeled either by labeling theprimer, or by incorporation of labeled nucleotides by the polymerase.Labels of use according to this embodiment of the invention include, butare not limited to fluorescent moieties, radioactive moieties, biotin,and digoxigenin.

Enrichment may also be enhanced by annealing and extending a primercomplementary to the original extended primer and repeating theextension steps. The oligonucleotide primer may also have an additional3′-terminal extension immediately adjacent to the sequence complementaryto the selected sequence. This extension, which may be one, two, three,on up to eight nucleotides or more beyond the sequence complementary tothe selected sequence, will effect further reduction in the complexityof the population when the primers are extended in the following steps.

3) Detect a Sequence Difference with Respect to a Reference Sequence.

Detection of sequence differences may be accomplished using any of themethods described in Examples 3 or 4, or elsewhere herein, or as knownin the art.

Example 8 Enriching for and Detecting a Nucleic Acid Sequence Differencewith Respect to a Reference Sequence

In this example, a nucleic acid sample is fragmented and a subset offragments is physically separated on the basis of their size. To performthe method, one must perform the following steps.

1) Fragment a Nucleic Acid Sample from One or More Individuals.

Nucleic acids may be fragmented by any of the methods discussed above.

2) Physically Separate a Subset of the Fragments Based on their Size.

Physical separation of nucleic acid fragments by size may beaccomplished in several different ways. First, electrophoreticseparation on a gel matrix may be performed according to standardmethods using agarose or polyacrylamide gel electrophoresis (see Ausubelet al., supra, pp. 2-13 and 2-23).

Second, fragments may be separated based on their position in a densitygradient. CsCl density gradient ultracentrifugation of nucleic acids isa standard method well known in the art. Also, the rate of migration ofDNA in a high-density sucrose gradient will vary with the size of thefragment (see, for example, Schans et al, 1969, Anal. Biochem., 32: 14).This is not a function of the density of the DNA, but of the size of theDNA and the effects of viscosity on migration. One may establish agradient by centrifugation, remove fractions with a fraction collector,and purify nucleic acids of a desired size (evaluated by electrophoresisof a sample from a fraction alongside nucleic acid standard markers).

3) Optionally Linking the Subset of Fragments Isolated on the Basis oftheir Size to Molecules Facilitating the Replication of the LinkedSubset.

Linkage may be performed by annealing and/or ligation of the subset ofmolecules isolated in step (2) to either a plasmid or to anoligonucleotide as described above in Example 4, section 2.

4) Replicate the Subset of Fragments Linked in Step (3) to Form anEnriched Collection of Replicated Molecules.

Replication may be performed in the same manner as replication of thesubset of nucleic acid molecules performed in Example 3.

5) Detect One or More Sequence Differences in the Members of theEnriched Collection Generated in Step (4) with Respect to a ReferenceSequence.

Detection of sequence differences is performed according to the samemethods as described for Example 8.

Example 9 Accessing a Sub-Portion of a Nucleic Acid Population

In this example, oligonucleotide primers are used to access asub-portion of a nucleic acid population in order to reduce thecomplexity of the population and facilitate subsequent analysis (e.g.,identification of polymorphisms). An advantage of this method is that itallows reproducible access to a given sub-portion of nucleic acidmolecules from the same individual and from different individuals withina given population. To access a sub-portion of a nucleic acid populationaccording to this aspect of the invention, one must perform thefollowing steps.

1. Anneal One or More Oligonucleotide Primers with a Sample of NucleicAcid.

The oligonucleotide primers used comprise a 3′-terminal sequencecomplementary to a selected sequence present in the nucleic acidmolecules of the sample. The length of the sequence may be varieddepending on the size of the sub-portion of the sequences one wishes toaccess, but will generally be at least about 5 nt in length or longer.The sequence may correspond to any sequence known or predicted to occurin the molecules of the nucleic acid sample. In addition to the sequencecomplementary to a selected sequence, the oligonucleotide primer mayhave additional nucleotides 5′ of the selected sequence that willfacilitate subsequent analysis steps.

The oligonucleotide primer may also have an additional 3-terminalextension immediately adjacent to the sequence complementary to theselected sequence. This extension, which may be one, two, three, on upto eight nucleotides or more beyond the sequence complementary to theselected sequence, will effect further reduction in the complexity ofthe population when the primers are extended in the following steps.

According to this embodiment of the invention, the oligonucleotideprimers may additionally be attached to a solid support or be labeledwith a moiety allowing attachment to a solid support. Methods forattaching oligonucleotides to solid supports are known in the art. Oneskilled in the art may determine the annealing conditions for a givenoligonucleotide primer or primers in this method (see Example 3). Theconditions for annealing will depend on the length and G+C content ofthe hybrid comprising the selected sequence and its complement in theoligonucleotide primer, plus any 3′ terminal extension, and on the saltconcentration of the buffer used. Generally, the salt concentration willcorrespond to the optimal concentration for the template-dependentpolymerase chosen for the primer extension step.

2) Extend the Annealed Primer to Generate a Population Comprising aSub-Portion of the Nucleic Acid Molecules in the Sample.

Extension of the annealed oligonucleotide primers is performed using atemplate-dependent polymerase such as Taq DNA polymerase or Klenow DNApolymerase under conditions either as specified by the enzyme supplieror as modified by one of skill in the art. Under certain circumstances(e.g., when one wishes to further limit the complexity of the resultingpopulation), one may add a chain-terminating nucleoside analog to theextension mixture at a concentration that limits the length of theaverage extension product. Within this embodiment of the invention, onemay wish to limit the length of the average extension product to anylength between about 500 and 5000 nt. One of skill in the art maydetermine the concentration of chain-terminating nucleoside analog toadd to achieve a given desired average extension product length with aminimum of experimentation.

The extension products may be detectably labeled either by labeling theprimer, or by incorporation of labeled nucleotides by the polymerase.Labels of use according to this embodiment of the invention include, butare not limited to fluorescent moieties, radioactive moieties, biotin,and digoxigenin.

The sub-portion of the nucleic acid population accessed according tothis embodiment of the invention represent a population of reducedcomplexity that may then be used to identify a nucleic acid sequencepolymorphism in a population or in an individual using methods asdescribed elsewhere herein.

Example 10 Accessing a Sub-Population of a Genome

In this example, a sub-population of a genome is accessed in order toreduce the complexity of the genome for subsequent analyses. Accordingto this aspect of the invention, one must take the following steps.

1) Cleave a Nucleic Acid Sample with One or More Cleavage Agents.

The Cleavage agent or agents may be sequence-specific cleavage agents,and will preferably cleave infrequently in the genome. Cleavage with asequence-specific cleavage agent may be performed as described inExample 4, as described elsewhere herein, or in a manner known in theart for a given cleavage agent.

2) Link an Oligonucleotide to the Ends Generated by theSequence-Specific Cleavage Agent.

Linkage may be by annealing, or by ligation or both. In the case wherelinkage is by annealing, this step involves addition of eithersingle-stranded oligonucleotides or double-stranded oligonucleotideswith a single-stranded overhang capable of annealing to the endsgenerated by the cleavage agent. It is possible to achieve extension ofan oligonucleotide annealed but not ligated to a fragment by way of anoverhang.

When an oligonucleotide is ligated, it will be a double-strandedoligonucleotide adapter with an overhang capable of annealing to thefragment ends generated by the cleavage agent. In some instances theannealed oligonucleotide may regenerate the sequence recognized by thesequence-specific cleavage agent. It is also possible to ligate anoligonucleotide adapter comprising a free end or nick capable of beingextended by a strand-displacing polymerizing activity. It is alsopossible to ligate an adapter comprising a sequence capable of beingnicked (e.g., an adapter with a mismatched bulge susceptible to cleavageby an enzyme, such as S1 nuclease, that cleaves at mismatched bases).

3) Extend the Oligonucleotide Linked in Step (2).

Extension may be achieved, as noted in step (2) by addition of a nucleicacid polymerizing activity and nucleotides under conditions favored forthe particular polymerizing activity used.

Alternatively, extension may be achieved by annealing a single-strandedoligonucleotide complementary to an oligonucleotide ligated in step (2),or complementary to the sequence-specific cleavage agent siteregenerated by the ligated sequence, and adding a nucleic acidpolymerizing activity and nucleotides under conditions favored for theparticular polymerizing activity used. Nucleic acid polymerizingactivities may include any template-dependent polymerizing activity,such as, without limitation, Klenow DNA polymerase, Taq DNA polymerase,or an RNA polymerase such as Sp6, T7 or T3 RNA polymerase. In the caseof RNA polymerases, the oligonucleotide ligated to the cleaved fragmentsmust comprise a promoter sequence for the selected RNA polymerase.

The extension may be repeated to increase the enrichment of sequences.

In order to generate an enriched sub-portion of the genome by thismethod, the extension must be limited to avoid the theoreticalreplication of the entire genome, which would not enrich for sequencesnear the sites recognized by the sequence-specific cleavage agent. Oneway to limit the length of the extension products is to include a chosenconcentration of chain-terminating nucleotide analogs (such asdideoxynucleotides) to the extension mix. For example, one may addenough of a dideoxynucleotide to limit the average extension product toabout 500 nt, 750 nt, 1000 nt, 1500 nt, 2000 nt, 3000 nt, 4000 nt, oreven about 5000 nt. For a sequence-specific cleavage agent that gives anaverage fragment size of 10,000 base pairs or more, this will result inreplication of less than half the sequence of the average fragment. Thatis, the inclusion of one or more chain terminating nucleotide analogswill result in the generation of an incomplete extension product.

Another aspect of this method that will further reduce the complexity ofthe nucleic acid molecule population is the use of a primer that has a3′-terminal extension immediately adjacent to the cleavage agentrecognition site. This extension, which may be one, two, three, on up toeight nucleotides or more beyond the sequence complementary to thesequence recognized by the cleavage agent, will effect a furtherreduction in the complexity of the population when the primers areextended. The reduction in complexity effected by the inclusion of 3′terminal extensions on a primer is proportional to the length of the3′-terminal extension; the longer the extension, the greater thereduction in complexity.

Nucleic acid of reduced complexity generated according to this methodmay be further analyzed to identify polymorphisms in individuals or in apopulation of individuals using methods described herein or as known inthe art.

1-35. (canceled)
 36. A process that can be used to identify in a nucleicacid sample the presence or absence of nucleic acid sequencedifferences, wherein each said difference is with respect to a referencesequence, the process comprising: a. fragmenting nucleic acids in saidsample; b. ligating adapter sequences to the nucleic acid fragmentsgenerated in step a; c. replicating said adapter linked nucleic acidfragments of step b; d. binding said replicated and adapter ligatednucleic acid fragments to a solid support by hybridizingoligonucleotides attached to the solid support to the adapter sequences;and e. identifying nucleic acid sequences within said replicated andsolid support bound nucleic acid fragments by hybridizing primers to thebound and replicated fragments and ligating the primers tooligonucleotides which are also hybridized to the replicated and solidsupport bound fragments.
 37. The process of claim 1 wherein steps b andc are repeated at least once prior to binding to the solid support instep d.
 38. A process to identify in a nucleic acid sample the presenceor absence of nucleic acid sequence differences, wherein each saiddifference is with respect to one or more reference sequences, theprocess comprising: a. contacting a nucleic acid sample, or a replicathereof, with molecules comprising sequence-specific binding activityunder conditions which permit binding; b. operatively linking saidmolecules comprising sequence-specific binding activity to one anotherwhen said molecules bind to the nucleic acid sample, or a replicathereof, under conditions which permit said molecules to be operativelylinked; c. replicating the operatively linked molecules of step b; d.binding the replicated molecules of step c to a solid support to formbound replicated molecules; and e. identifying nucleic acid sequenceswithin said replicated molecules.
 39. A process of claim 38 whereinoperatively linking said molecules comprising sequence-specific bindingactivity in step b, is achieved by ligating said molecules.
 40. Aprocess of claim 38 wherein operatively linking said moleculescomprising sequence-specific binding activity in step b is achieved bymodifying one or more said molecules with a polymerase.
 41. A process ofclaim 38 whereby the replicating in step c includes amplification usinga molecule with polymerase activity.
 42. A process of claim 38 whereinthe bound replicated molecules of step d, are amplified on the solidsupport using a molecule with polymerase activity.
 43. A process ofclaim 38 whereby the operatively linked molecules comprisingsequence-specific binding activity of step b are replicated in step cusing primers with adapter sequences.
 44. A process of claim 43 whereinthe molecules replicated using primers with adapter sequences are boundto the solid support using said adapter sequences.
 45. A process toidentify in a nucleic acid sample the presence or absence of nucleicacid sequence differences, wherein each said difference is with respectto one or more reference sequences, the process comprising: a.contacting a nucleic acid sample, or a replica thereof, with moleculescomprising sequence-specific binding activity under conditions whichpermit binding; b. operatively linking said molecules comprisingsequence-specific binding activity to one another when said moleculesbind to the nucleic acid sample, or a replica thereof, under conditionswhich permit said molecules to be operatively linked; c. binding theoperatively linked molecules to a solid support and replicating saidbound molecules; and d. identifying nucleic acid sequences within saidreplicated molecules.
 46. A process of claim 45 wherein operativelylinking said molecules comprising sequence-specific binding activity instep b, is achieved by ligating said molecules.
 47. A process of claim45 wherein operatively linking said molecules comprisingsequence-specific binding activity in step b is achieved by modifyingone or more said molecules with a polymerase.
 48. A process of claim 45whereby the replicating in step c includes amplification using amolecule with polymerase activity.
 49. A process of claim 45 wherein thebound operatively linked molecules of step c, are replicated on thesolid support using amplification with a molecule with polymeraseactivity.
 50. A process of claim 45 whereby the operatively linkedmolecules comprising sequence-specific binding activity of step b arereplicated in step c using primers with adapter sequences before bindingsaid molecules to the solid support.
 51. A process of claim 50 whereinthe molecules replicated using primers with adapter sequences are boundto the solid support using said adapter sequences.
 52. A process toidentify in a nucleic acid sample the presence or absence of nucleicacid sequence differences, wherein each said difference is with respectto one or more reference sequences, the process comprising: a.contacting a nucleic acid sample, or a replica thereof, with moleculescomprising sequence-specific binding activity under conditions whichpermit binding; b. operatively linking said molecules comprisingsequence-specific binding activity to one another when said moleculesbind to the nucleic acid sample, or a replica thereof, under conditionswhich permit said molecules to be operatively linked; c. replicating theoperatively linked molecules of step b; d. binding the replicatedmolecules of step c to a solid support to form bound replicatedmolecules; and e. identifying nucleic acid sequences within said boundand replicated molecules.
 53. A process of claim 52 wherein operativelylinking said molecules comprising sequence-specific binding activity instep b, is achieved by ligating said molecules.
 54. A process of claim52 wherein operatively linking said molecules comprisingsequence-specific binding activity in step b is achieved by modifyingone or more said molecules with a polymerase.
 55. A process of claim 52whereby the replicating in step c includes amplification using amolecule with polymerase activity.
 56. A process of claim 52 wherein thebound replicated molecules of step d, are amplified on the solid supportusing a molecule with polymerase activity.
 57. A process of claim 52whereby the operatively linked molecules comprising sequence-specificbinding activity of step b are replicated in step c using primers withadapter sequences.
 58. A process of claim 57 wherein the moleculesreplicated using primers with adapter sequences are bound to the solidsupport using said adapter sequences.
 59. A process to identify in anucleic acid sample the presence or absence of nucleic acid sequencedifferences, wherein each said difference is with respect to one or morereference sequences, the process comprising: a. contacting a nucleicacid sample, or a replica thereof, with molecules comprisingsequence-specific binding activity under conditions which permitbinding; b. operatively linking said molecules comprisingsequence-specific binding activity to one another when said moleculesbind to the nucleic acid sample, or a replica thereof, under conditionswhich permit said molecules to be operatively linked; c. binding theoperatively linked molecules to a solid support and replicating saidbound molecules; and d. identifying nucleic acid sequences within saidbound and replicated molecules.
 60. A process of claim 59 whereinoperatively linking said molecules comprising sequence-specific bindingactivity in step b, is achieved by ligating said molecules.
 61. Aprocess of claim 59 wherein operatively linking said moleculescomprising sequence-specific binding activity in step b is achieved bymodifying one or more said molecules with a polymerase.
 62. A process ofclaim 59 whereby the replicating in step c includes amplification usinga molecule with polymerase activity.
 63. A process of claim 59 whereinthe bound operatively linked molecules of step c, are replicated on thesolid support using amplification with a molecule with polymeraseactivity.
 64. A process of claim 59 whereby the operatively linkedmolecules comprising sequence-specific binding activity of step b arereplicated in step c using primers with adapter sequences before bindingsaid molecules to the solid support.
 65. A process of claim 64 whereinthe molecules replicated using primers with adapter sequences are boundto the solid support using said adapter sequences.